Vertical‐scanning mutagenesis of amino acids in a model N‐myristoylation motif reveals the major amino‐terminal sequence requirements for protein N‐myristoylation

Abstract

In order to determine the amino-terminal sequence requirements for protein N-myristoylation, site-directed mutagenesis of the N-terminal region was performed using tumor necrosis factor (TNF) mutants as model substrate proteins. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system using rabbit reticulocyte lysate. A TNF mutant having the sequence MGAAAAA AAA at its N-terminus was used as the starting sequence to identify elements critical for protein N-myristoylation. Sequential vertical-scanning mutagenesis of amino acids at a distinct position in this model N-terminal sequence revealed the major sequence requirements for protein N-myristoylation: the combination of amino acids at position 3 and 6 constitutes a major determinant for the susceptibility to protein N-myristoylation. When Ser was located at position 6, 11 amino acids (Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His) were permitted at position 3 to direct efficient protein N-myristoylation. In this case, the presence of Lys at position 7 was found to affect the amino acid requirement at position 3 and Lys became permitted at this position. When Ser was not located at position 6, only 3 amino acids (Ala, Asn, Gln) were permitted at position 3 to direct efficient protein N-myristoylation. The amino acid requirements found in this study were fully consistent with the N-terminal sequence of 78 N-myristoylated proteins in which N-myristoylation was experimentally verified. These observations strongly indicate that the combination of amino acids at position 3, 6 and 7 is a major determinant for protein Nmyristoylation.

Machine Learning Prediction of Amino Acid Patterns in Protein N-myristoylation

“…8 and 10. Although one rule is a known fact confirmed by the biological experiment [2], the other rule suggests new amino acid sequence patterns for N-myristoylation.…”

“…As shown in Fig. 3, when Ser is located at position 6, 11 amino acid residues (Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His) are permitted locating at position 3 to direct efficient protein N-myristoylation [2,3]. Most of these 11 amino acids have a rule that the radius of gyration of residue is smaller than 1.80Å.…”

“…9 summarizes a relationship between the amino acid pattern dependency at the position 3 on Ser at position 6 and the result of indexing from BONSAI. Eleven amino acid residues, which are biologically determined to be located at position 3 under the existence of Ser at the position 6 [2], are marked with black circles in the figure. By comparing the black circles pattern and the result of indexing, we can see that, out of these 11 amino acid residues, 9 amino acid residues (except Val and Gln) have been classified to the symbol 0.…”

“…In addition to the amino acids at position 6, there is a case that some amino acid residues at position 7 affects amino acid requirement at position 3 for N-myristoylation. For example, although location of Ser at position 6 does not basically allow Lys to locate at position 3, location of Lys at position 7 makes a changes to the requirement for amino acid residue at position 3; Lys can be located at position 3 [2].…”

“…In order to determine the amino-terminal sequence requirements for protein N-myristoylation, their sequences have been examined [2,3]. Most of methods used by researchers are those that predict patterns for N-myristoylation by biological experimentations based on their knowledge.…”