Structural basis for intramolecular interaction of post-translationally modified H-Ras•GTP prepared by protein ligation
Ke, H.; Matsumoto, S.; Murashima, Y.; Taniguchi-Tamura, H.; Miyamoto, R.; Yoshikawa, Y.; Tsuda, C.; Kumasaka, T.; Mizohata, E.; Edamatsu, H.; Kataoka, T.
Febs Letters 591(16): 2470-2481
2017
ISSN/ISBN: 0014-5793 PMID: 28730604 DOI: 10.1002/1873-3468.12759
Accession: 060283722
Ras undergoes post-translational modifications including farnesylation, proteolysis, and carboxymethylation at the C terminus, which are necessary for membrane recruitment and effector recognition. Full activation of c-Raf-1 requires cooperative interaction of the farnesylated C terminus and the activator region of Ras with its cysteine-rich domain (CRD). However, the molecular basis for this interaction remains unclear because of difficulties in preparing modified Ras in amounts sufficient for structural studies. Here, we use Sortase A-catalyzed protein ligation to prepare modified Ras in sufficient amounts for NMR and X-ray crystallographic analyses. The results show that the farnesylated C terminus establishes an intramolecular interaction with the catalytic domain and brings the farnesyl moiety to the proximity of the activator region, which may be responsible for their cooperative recognition of c-Raf-1-CRD.