Ben Douglass (1),
Aaron Downs (3) and David
© David Marcey, 2001
III. HhaI DNA Methyltransferase Monomer Structure
IV. Binding Mechanism
V. HhaI DNA Methyltransferase Complexed with Cofactor: AdoMet binding
VI. HhaI DNA Methyltransferase Complexed with DNA and AdoMet
Note: This exhibit is best viewed if the cue buttons ( ) are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.
Methyltransferases recognize specific DNA sequences and transfer a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to nitrogenous bases. Methyltransferases are employed in restriction-modification and mismatch repair systems in prokaryotes, and have been implicated in many molecular processes in eukaryotes including regulation of gene expression, genomic imprinting, DNA repair, mutagenesis, and chromatin organization.
There are three classes of methyltransferases. Two of the classes methylate exocyclic nitrogens to convert adenine to N6-methyladenine and cytosine to N4-methylcytosine. The third class methylates the fifth cytosine carbon to convert it to 5-methylcytosine; this class is referred to as m5c-methyltransferases. All family members of m5c-methyltransferases are built upon a common architecture of ten conserved motifs (conserved blocks of amino acids). The majority of these conserved, structural motifs are located on the surface of the binding cleft of the molecule.
The reaction mechanism of m5c-methyltransferases begins with a nucleophilic attack by a cysteine residue (Cys81) on the sixth cytosine carbon of the targeted cytosine base. This attack creates a covalent intermediate that serves to activate the fifth cytosine carbon as a carbanion that is stabilized by resonance. The carbanion attacks the methyl group of AdoMet nucleophilically, which causes the addition of the methyl group to the fifth carbon. AdoMet is altered to AdoHcy after the methyl group is removed by the carbanion. There is a concerted disengagement of the cysteine residue from the sixth cytosine carbon, addition of a hydrogen ion to the cysteine residue, and the formation of a double bond between the fifth and sixth cytosine carbons.
HhaI DNA methyltransferase
is a m5c-methyltransferase that recognizes the 5'-GCGC-3' sequence in double stranded
DNA and methylates the first cysteine of the recognition sequence. The molecule
is comprises 327 amino acids and has dimensions of 40 Å x 50 Å x 60
Å. HhaI has three domains, the large domain
and small domain, connected by a hinge
region . Note the pronounced cleft between the large domain
and small domain. This cleft is capable of binding
double stranded B-DNA (see below ).
The large domain contains a core b-sheet
with six strands (five shown in ribbons) . Four of the strands are parallel to one another, and the remaining
two strands form a hairpin b
sheet turn at one end of the four parallel strands. The core
complex is sandwiched between two
a helices and two a
helices plus one b
strand . There is also an a
helix that lies in front of the b
sheet core . The large domain contains all or parts
of ten of the conserved motifs found in m5c methyltransferases.
The small domain contains seven
strands . Five b
strands are configured in an antiparallel formation and are arranged
in a circular formation that resembles a pinwheel. There are two
strands above and below the antiparallel group, orientated in opposite
The hinge region is composed of an a-b-a structure that connects
the large and small domains and includes parts of conserved motifs IX and X.
The first half of motif IX is in the small domain, and provides much of the
structural backbone for the small domain. The second
half of motif X is located in the large domain.
The large domain contains a core b-sheet with six strands (five shown in ribbons) . Four of the strands are parallel to one another, and the remaining two strands form a hairpin b sheet turn at one end of the four parallel strands. The core b-sheet complex is sandwiched between two a helices and two a helices plus one b strand . There is also an a helix that lies in front of the b sheet core . The large domain contains all or parts of ten of the conserved motifs found in m5c methyltransferases.
The small domain contains seven b strands . Five b strands are configured in an antiparallel formation and are arranged in a circular formation that resembles a pinwheel. There are two strands above and below the antiparallel group, orientated in opposite directions.
The hinge region is composed of an a-b-a structure that connects the large and small domains and includes parts of conserved motifs IX and X. The first half of motif IX is in the small domain, and provides much of the structural backbone for the small domain. The second half of motif X is located in the large domain.
The amino acid sequences between motifs VIII and IX are not conserved in all members of m5c-methyltransferases and are labeled the variable region . This region has the greatest heterogeneity in size, sequence, and composition among family members. The variable region spans the entire length of the HhaI molecule and folds to form the majority of the small domain. There is a small sub region within the variable region that is responsible for sequence specific recognition and target base selection. The target recognition domains are located on the side of the cleft in the small domain and directly interact with the major groove of the DNA molecule.
The HhaI DNA methyltransferase molecule has two seperate binding pockets for DNA and AdoMet. The pockets are in close proximity, and allow the three molecules to interact. HhaI binds DNA, bringing about structural changes that enables the binding of AdoMet to form a tertiary complex.
This is HhaI DNA Methyltransferase structure when complexed with AdoMet .The binding of AdoMet by HhaI methyltransferase is unique. The AdoMet binding site is located in the large domain. The site is a hydrophobic pocket bounded by the a helix in front of the b sheet core and by five residues in four conserved motifs : motif I, motif III, motif IV, and motif X. Other residues surrounding the AdoMet binding pocket interact with the cofactor to tightly bind it to the protein . These residues (partially conserved or barely conserved) use a variety of hydrogen bonds and side chain interactions to accomplish the binding of the cofactor. AdoMet is inserted into the pocket so that the methionine projects into the cleft. There is a glycine rich loop inside the binding pocket that serves to position the AdoMet molecule so that the methionine region is in close proximity with the DNA .
This is the HhaI DNA Methyltransferase structure when complexed with DNA and AdoMet. DNA binds to HhaI methyltransferase in the binding cleft formed by the three domains and is situated so that the major groove faces the small domain and the minor groove faces the large domain. There are three sites of protein/DNA interaction: two glycine rich loops in the small domain and the active site loop in the large domain . The HhaI monomer conformation is altered by DNA binding. This change brings the active site loop towards the binding cleft, where it contacts the minor groove of DNA and binds in three places . The binding of DNA also brings the small domain closer to the binding cleft so that the glycine rich loops can come into contact with the major groove of the DNA molecule .
In order for the HhaI molecule
to methylate the cytosine base, the DNA must undergo structural distortions.
Commonly, structural distortions caused by DNA binding proteins result in DNA
bends. These proteins usually contact the surface of bases in the major or minor
grooves and interact with the external, phosphodiester backbone. In the case
of HhaI, however, the buried atoms of cytosine bases of DNA must be accessible
for methylation chemistry, requiring drastic distortions to make the bases accessible
to the methyltransferase. The phosphodiester backbone of DNA is distorted in
such a manner that the phosphates on either side of the target cytosine are
shifted outward, increasing the distance between the phosphates on the two strands
of DNA. The shifting of the phosphates allows the target cytosine
to flip out of the DNA helix through the minor groove . Once out of the helix, the target cytosine is held in place by three
conserved motifs surrounding the binding cleft in the large domain using
hydrogen bonds and salt bridges . A simple removal of the target cytosine would be enrgetically
unfavorable, and two residues located on two
separate loops move into the helix to occupy the position vacated by
the target cytosine, thus restoring stacking interactions
. These residues are unique to HhaI methyltransferase.
The conformational changes bring the target cytosine,
the catalytic nucleophile of the active site loop (cysteine81),
and the methyl donor (AdoMet) into close proximity
Cheng, X., Kumar, S., Posfai,P.,Pflugrath, J., Roberts, R.(1993). Crystal Structure of the HhaI DNA Methyltransferase Complexed with S-Adenosyl-L-Methionine. Cell. 74:299-307.
Klimasauskas, S., Kumar, S., Roberts, R., Cheng, X.(1994). HhaI Methyltransferase Flips Its Target Base Out of the DNA Helix. Cell. 76: 357-369.