Myristoylation is a lipidation modification where a myristoyl group, derived from myristic acid, is covalently attached by an amide bond to the alpha-amino group of an N-terminal glycine residue. Myristic acid is a 14-carbon saturated fatty acid (14:0) with the systematic name of n-tetradecanoic acid. This modification can be added either co-translationally or post-translationally. N-myristoyltransferase (NMT) catalyzes the myristic acid addition reaction in the cytoplasm of cells. This lipidation event is the most common type of fatty acylation and is present in many organisms, including animals, plants, fungi, protozoans and viruses. Myristoylation allows for weak protein–protein and protein–lipid interactions and plays an essential role in membrane targeting, protein–protein interactions and functions widely in a variety of signal transduction pathways.
Discovery In 1982, Koiti Titani's lab identified an "N-terminal blocking group" on the catalytic subunit of cyclic AMP-dependent protein kinase in cows as n-tetradecanoyl. Almost simultaneously in Claude B. Klee's lab, this same N-terminal blocking group was further characterized as myristic acid. Both labs made this discovery utilizing similar techniques: mass spectrometry and gas chromatography.
N-myristoyltransferase
The enzyme N-myristoyltransferase (NMT) or glycylpeptide N-tetradecanoyltransferase is responsible for the irreversible addition of a myristoyl group to N-terminal or internal glycine residues of proteins. This modification can occur co-translationally or post-translationally. In vertebrates, this modification is carried about by two NMTs, NMT1 and NMT2, both of which are members of the GCN5 acetyltransferase superfamily.
Structure The crystal structure of NMT reveals two identical subunits, each with its own myristoyl CoA binding site. Each subunit consists of a large saddle-shaped β-sheet surrounded by α-helices. The symmetry of the fold is pseudo twofold. Myristoyl CoA binds at the N-terminal portion, while the C-terminal end binds the protein.
Mechanism The addition of the myristoyl group proceeds via a nucleophilic addition-elimination reaction. First, myristoyl coenzyme A (CoA) is positioned in its binding pocket of NMT so that the carbonyl faces two amino acid residues, phenylalanine 170 and leucine 171. This polarizes the carbonyl so that there is a net positive charge on the carbon, making it susceptible to nucleophilic attack by the glycine residue of the protein to be modified. When myristoyl CoA binds, NMT reorients to allow binding of the peptide. The C-terminus of NMT then acts as a general base to deprotonate the NH3+, activating the amino group to attack at the carbonyl group of myristoyl-CoA. The resulting tetrahedral intermediate is stabilized by the interaction between a positively charged oxyanion hole and the negatively charged alkoxide anion. Free CoA is then released, causing a conformational change in the enzyme that allows the release of the myristoylated peptide.
Co-translational vs. post-translational addition Co-translational and post-translational covalent modifications enable proteins to develop higher levels of complexity in cellular function, further adding diversity to the proteome. The addition of myristoyl-CoA to a protein can occur during protein translation or after. During co-translational addition of the myristoyl group, the N-terminal glycine is modified following cleavage of the N-terminal methionine residue in the newly forming, growing polypeptide. Post-translational myristoylation typically occurs following a caspase cleavage event, resulting in the exposure of an internal glycine residue, which is then available for myristic acid addition.
Functions
Myristoylated proteins
Myristoylation molecular switch Myristoylation not only diversifies the function of a protein, but also adds layers of regulation to it. One of the most common functions of the myristoyl group is in membrane association and cellular localization of the modified protein. Though the myristoyl group is added onto the end of the protein, in some cases it is sequestered within hydrophobic regions of the protein rather than solvent exposed. By regulating the orientation of the myristoyl group, these processes can be highly coordinated and closely controlled. Myristoylation is thus a form of "molecular switch." Both hydrophobic myristoyl groups and "basic patches" (highly positive regions on the protein) characterize myristoyl-electrostatic switches. The basic patch allows for favorable electrostatic interactions to occur between the negatively charged phospholipid heads of the membrane and the positive surface of the associating protein. This allows tighter association and directed localization of proteins. Myristoyl-conformational switches can come in several forms. Ligand binding to a myristoylated protein with its myristoyl group sequestered can cause a conformational change in the protein, resulting in exposure of the myristoyl group. Similarly, some myristoylated proteins are activated not by a designated ligand, but by the exchange of GDP for GTP by guanine nucleotide exchange factors in the cell. Once GTP is bound to the myristoylated protein, it becomes activated, exposing the myristoyl group. These conformational switches can be utilized as a signal for cellular localization, membrane-protein, and protein–protein interactions.
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