RasIns: Genetically Encoded Intrabodies of Activated Ras Proteins

• Published in: Journal of molecular biology 2017-02, Vol.429 (4), p.562-573
• Main Authors: Cetin, Mehmet, Evenson, William E., Gross, Garrett G., Jalali-Yazdi, Farzad, Krieger, Daniel, Arnold, Don, Takahashi, Terry T., Roberts, Richard W.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies - genetics; Antibodies - metabolism; Chlorocebus aethiops; COS Cells; E10FnIII; fibronectin; Guanosine Diphosphate - metabolism; Guanosine Triphosphate - metabolism; High-Throughput Nucleotide Sequencing; Humans; intrabody; mRNA display; Mutation; Protein Conformation; Ras; ras Proteins - genetics; ras Proteins - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Signal Transduction
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2016.11.008

K- and H-Ras are the most commonly mutated genes in human tumors and are critical for conferring and maintaining the oncogenic phenotype in tumors with poor prognoses. Here, we design genetically encoded antibody-like ligands (intrabodies) that recognize active, GTP-bound K- and H-Ras. These ligands, which use the 10th domain of human fibronectin as their scaffold, are stable inside the cells and when fused with a fluorescent protein label, the constitutively active G12V mutant H-Ras. Primary selection of ligands against Ras with mRNA display resulted in an intrabody (termed RasIn1) that binds with a KD of 2.1μM to H-Ras(G12V) (GTP), excellent state selectivity, and remarkable specificity for K- and H-Ras. RasIn1 recognizes residues in the Switch I region of Ras, similar to Raf-RBD, and competes with Raf-RBD for binding. An affinity maturation selection based on RasIn1 resulted in RasIn2, which binds with a KD of 120nM and also retains excellent state selectivity. Both of these intrabodies colocalize with H-Ras, K-Ras, and G12V mutants inside the cells, providing new potential tools to monitor and modulate Ras-mediated signaling. Finally, RasIn1 and Rasin2 both display selectivity for the G12V mutants as compared with wild-type Ras providing a potential route for mutant selective recognition of Ras. [Display omitted] •We used mRNA display and an e10FnIII scaffold to find state-specific binders of Ras: RasIn1 and RasIn2.•Rasin1 and RasIn2 are stable and functional inside the cells.•RasIn1 and RasIn2 bind K-Ras, H-Ras, and oncogenic mutants, but not Rap1B or Arf1 homologs.•RasIn2 has similar affinity for Ras as the Ras-binding domain of Raf1, the canonical binding partner of Ras.