Protein Synthesis DOI: 10.1002/anie.201001884 Synthesis of the Rheb and K-Ras4B GTPases** Yong-Xiang Chen, Sebastian Koch, Katharina Uhlenbrock, Katrin Weise, Debapratim Das, Lothar Gremer, Luc Brunsveld, Alfred Wittinghofer, Roland Winter, Gemma Triola, and Herbert Waldmann* Dedicated to Prof. Horst Kunz on the occasion of his 70th birthday. Small GTPases of the Ras superfamily critically regulate numerous cellular programs and are involved in the establish- ment of disease, in particular cancer. [1] Two relevant examples are the K-Ras4B protein and the Ras homologue enriched in brain (Rheb) protein. Rheb activates the mTORC1 (mam- malian target of rapamycin complex 1) signaling pathway, which plays a central role in regulating cell growth, prolifer- ation, and metabolism. [2] Increasing evidence suggests that Rheb and mTORC1 are aberrantly activated in a variety of human cancers. [3] The mechanism of mTORC1 activation by Rheb is subject to intense debate. [4] K-Ras4B is the most important isoform of the Ras proteins, which hold a central position in the transduction of growth-promoting signals across the plasma membrane to regulate cell growth and differentiation. Mutations in Ras that lead to misregulated signaling are found in approximately 30 % of all human cancers. [5] Both Rheb and K-Ras4B contain an S-farnesylated cysteine methyl ester at their C terminus (see Scheme 1). This posttranslational modification is required for their biological function. Thus, Rheb needs to be farnesylated and methylated for correct localization to endomembranes and for the activation of mTOR kinase, [3, 6, 7] and K-Ras4B needs to be S-farnesylated and carboxymethylated for selective localization to the plasma membrane and signaling activity. [8] For the study of the temporal and spatial organization of Rheb and K-Ras4B in cells, preparative amounts of fully posttranslationally modified and active protein, additionally equipped with suitable reporter groups and tags if required, would be invaluable. The production of such modified proteins by a combination of expression techniques and organic synthesis has proven to be an enabling technique for the study of the S-palmitoylated and S-farnesylated H- and N- Ras proteins and of the S-geranylgeranylated Rab pro- teins. [1, 9] However, farnesylated and carboxymethylated Rheb is not accessible by expression techniques, and, in contrast to the H- and N-Ras proteins, K-Ras4B has not succumbed to synthesis so far. In particular, the maleimide ligation that was very successful for the synthesis of H- and N-Ras [9] cannot be applied to K-Ras4B owing to the presence of multiple C- terminal lysine residues and the formation of inseparable product mixtures, and the use of the hydrazide linker employed previously in the synthesis of H- and N-Ras proteins resulted in only low yields of K-Ras4B peptides. [10] Herein we report the first synthesis of S-farnesylated Rheb and K-Ras4B methyl ester by a combination of expressed protein ligation (EPL) and lipopeptide synthesis. In EPL, a recombinant protein thioester generated by thiolysis of an intein fusion protein reacts with a synthetic peptide containing an N-terminal cysteine residue to yield a native amide bond. [11] For the efficient synthesis of Rheb by EPL, the Ala173 À Ala174 bond in the C-terminal flexible region [12] was chosen as the ligation site. Thus, a farnesylated peptide methyl ester 2 representing the Rheb 174–181 fragment in which Ala174 has been exchanged for Cys174 was required for the synthesis of Rheb, as well as a truncated RhebD11 thioester 3 (Scheme 1 a). Because of the acid sensitivity of the farnesyl group and to ensure that the C-terminal cysteine methyl ester was intro- duced without epimerization of the activated amino acid to which it needed to be attached, we anchored the peptide to an acid-sensitive trityl resin through the side-chain hydroxy group of Ser180 (Scheme 2). After selective removal of the allyl ester and coupling of the S-farnesylated cysteine methyl [*] Dr. Y. Chen, [+] Dr. S. Koch, [+] Dr. D. Das, Dr. G. Triola, Prof. Dr. H. Waldmann Abteilung Chemische Biologie Max-Planck-Institut für molekulare Physiologie Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) and Fachbereich Chemische Biologie, Fakultät Chemie Technische Universität Dortmund Otto-Hahn-Strasse 6, 44227 Dortmund (Germany) Fax: (+ 49) 231-133-2499 E-mail: herbert.waldmann@mpi-dortmund.mpg.de Dr. K. Uhlenbrock, [+] Dr. L. Gremer, Prof.Dr. A. Wittinghofer Abteilung Strukturelle Biologie Max-Planck-Institut für molekulare Physiologie (Germany) Dr. K. Weise, Prof. Dr. R. Winter Physikalische Chemie I Technische Universität Dortmund (Germany) Prof. Dr. L. Brunsveld Biomedical Engineering, Laboratory of Chemical Biology Eindhoven University of Technology (The Netherlands) [ + ] These authors contributed equally to this work. [**] This research was supported by the Max-Planck-Gesellschaft, the Fonds der Chemischen Industrie, the DFG (SFB 642), and the Alexander von Humboldt-Stiftung. We are grateful to Dr. C. Goemans and Prof. Dr. R. Heumann for providing DNA templates of Rheb, and C. Nowak for technical assistance. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201001884. Communications 6090  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2010, 49, 6090 –6095