Crystal Structure of Carnosine N-Methyltransferase bound to S-Adenosylmethionine (PDB ID: 5YF0) from Homo sapiens
Created by: Arthur Willmore
CARTNMT1 or carnosine N-methyltransferase 1 bound to
SAM (PDB ID: 5YF0) is a methyltransferase found in Homo sapiens that
methylates histidine at the N1 position of the imidazole ring. It is used in
biological recognition and regulation (1). CARNMT1 is part of the
methyl transferase family of proteins and more specifically is a member of the
class I methyltransferase proteins. The defining characteristic of
Class I methyltransferase proteins is their common substrate,
S-Adenosylmethionine (SAM) (2). Class I methyltransferase proteins
share a secondary structure known as a Rossman fold, which is conserved in SAM
binding methyltransferases. SAM acts as the methyl group donor in
Class I methyltransferases (2). Studying the structure and mechanism
of CARNMT1 can lead to a greater understanding of the effect of methylation as
a means of protein regulation as well as the mechanism via which substrate
recognition and protein specific catalysis occur (1).
CARNMT1like other methyltransferase proteins is
involved in post-translational modification of other proteins. The
primary method of action for class I methyltransferase proteins is SAM attacking
the enzyme substrate in a concerted Sn2 nucleophilic reaction. In
this nucleophilic attack, the methionine sulfur functions as the nucleophile
that transfers the methyl group to the enzyme substrate. CARNMT1 is
specifically a N1 methylation modifier, that exclusively methylates nitrogen at
the N1 position of a histidine imidazole ring. Due to the lack of
structural information for position specific transferases, the method of how
position specific recognition occurs is still poorly understood (1). Methylation
at the N1 position catalyzes the conversion of carnosine to anserine (1). Anserine
is a protein that is used in mammalian brain cells and skeletal muscles
cells. Protein methylation functions in a variety of ways
including, protein regulation, protein recognition, protein trafficking, signal
transduction and transcriptional regulation (3). Methyltransferases
can participate in protein regulation via epigenetic modification of the
genome. Methylation of histones in the genome can have the effect of
upregulating or downregulating DNA transcription (3). In experiments
to determine a crystal structure for CARNMT1 bound to SAM, researchers
determined protein regulation and protein recognition are two specific roles
for CARNMT1 bound to SAM in Homo sapiens (1).
CARNMT1 bound to SAM exists as a homodimer, with
the alpha and beta units of the protein being structurally identical (4). The
molecular weight of the protein is 86151.95 Da and the isoelectric point
is 5.58 (1,5). CARNMT1 has 4 ligands, acetate ion, calcium
ion, 1,2-ethanediol, and S-Adenosylmethionine (SAM), however the only ligand
with an explicitly known biological function is SAM (4).
The primary structure of CARNMT1 bound to SAM consists of 362
residues. Binding sites for the acetate, 1,2-ethanediol, and SAM
ligands have all been identified on the primary structure of CARNMT1. The
secondary structure of CARNMT1 consists of 15 helices comprised of 160 residues
that forms 44% of the subunit structure and 13 betas strands formed of 68
residues that make up 18% of the structure (4). Several elements of the
secondary structure form the binding pocket for carnosine, these include αD,
L4, L5, α5’ and L67. The Rossman fold is consists of
alternating beta strands and alpha helices, the beta strands for a plane near
the center of the protein and are bordered by alpha helices on each side of the
plane.
The CARNMT1 consists of two identical monomers, each
monomer binds a carnosine and SAM. The two carnosine molecules are
separated by a pair of tyrosine residues Tyr-396. Residues Tyr-396
and His-347 undergo induced side-chain rotamer change when carnosine is bound
in the pocket. In each subunit of CARNMT1 the conformation of the
Tyr-396 residue is identical, however when the CARNMT1 monomers dimerize to
form the quaternary structure, the two Tyr-396 residues will become disordered
and adopt distinct conformations. The purpose of this interaction
between the two residues is suspected to be a form of substrate
cross-talk. How this substrate cross-talk impacts the function of
the protein is currently unknown. The residues that directly form
the active carnosine pocket are Phe-313, Asp-316, Pro-343,
Leu-345, His-347, Tyr-386, Tyr-396 and Tyr-398. The orientation of
carnosine in the pocket is determined by hydrophobic interactions between
residues Phe-313, Pro-343, Leu-345 and Tyr-386 as well as hydrogen bonds
donated by resides Asp-316 and Tyr-398. Asp-316 hydrogen
bonds to the N3 of histidine which causes the residue and the imidazole ring of
histidine to flip so that the N1 position is adjacent to SAM. This
flipping of the imidazole is what causes the location specific methylation of
N1 on the histidine. After the methyl group has been donated from
SAM to the histidine, His-347 will flip back to its substrate free position.
When His-347 was mutated to His-347A and His-347F a more than 80% loss of
activity was observed which supports the necessity of His-347 to CARNMT1’s
mechanism (4).
Carnosine N-methyltransferase bound by AdoHcy (PDB
ID: 5X62) has a similar structure to CARNMT1 bound to SAM. Using DALI and
PSI-BLAST to test for similarity, Carnosine N-methyltransferase bound by AdoHcy
had a Z Score of 38.5 and an E value 1e-173 (6,7). CARNMT1 and
Carnosine N-methyltransferase bound by AdoHcy are both homodimers. They
are both carnosine methyltransferase proteins and have similar sequences and
secondary structures however Carnosine N-methyl transferase bound by AdoHcy is
44 residues longer (8). They also have different ligands and
substrates. The difference in ligands is most interesting
discrepancy between the two proteins. Although they use different
ligands both protein structures are similar and have similar functions. Carnosine
N-methyltransferase bound by AdoHcy has an S-Adenosyl-L-Homocysteine ligand
unlike CARNMT1. Whereas the CARNMT1 dimer binds two SAM ligands and
two carnosines, Carnosine N-methyltransferase bound by AdoHcy dimers bind one
SAM, one SAH, and two carnosines (2). This structural variation
between the two protein subunits led researchers to hypothesize that only half
of the dimer is functionally active. Since SAM is necessary for
methylation to occur and Carnosine N-methyltransferase bound by AdoHcy only has
one SAM across two subunits as opposed to CARNMT1 which has two only one of the
Carnosine N-methyltransferase bound by AdoHcy subunits is catalytically active
(2). Seeing as how location specific methylation occurs is still
poorly understood further exploration of how the two proteins differ could be
beneficial into understanding different mechanisms of substrate localized
reactions.
A second comparison was done using DALI and PSI-BLAST between
CARNMT1 and yeast Coq5 in the SAM bound form (PDB ID: 4OBW) (8). The
E value for yeast Coq5 in the SAM bound form was 0.78 and the z-score was 17.6
(6,7). Coq5, like CARNMT1, is a methyltransferase bound to the
ligand SAM however where CARNMT1 methylated the N3 position of histidine Coq5
is instead methylates C5 on coenzyme Q(9). Coq5 uses a similar
structure to CARNMT1 to form the active site for the substrate however because
Coq5 and CARNMT1 have different substrates the residues involved in forming the
active site are different. The active site for Coq5 is formed from
hydrophobic interactions between Tyr-78, Met-81, Tyr-263 and Leu-264 that fix
the substrate and Arg-77 and Asn-202 which form hydrogen bonds to further bind
the substrate (9). The SAM binding site is conserved between the two
proteins which supports the hypothesis of the role of SAM as a nucleophile in
the reaction (9).
Ultimately, the elegant structure of CARNMT1 bound to SAM
lends great insight into the mechanisms of location specific substrate
methylation. Without the unique mechanism and rotation of residue
His-347 the specific methylation of N1 on histidine would not be possible.
Further research into CARNMT1 and other methyltransferases suchs as Coq5 and
Carnosine N-methyl transferase bound by AdoHcy can help to gain a deeper
understanding of the role of SAM in biological processes and protein
methylation in general. Understanding the SAM ligand and protein
methylation has consequences for protein regulation, protein recognition, protein trafficking, signal
transduction and transcriptional regulation.