Genomic Sciences Center, RIKEN* Tohoku University Biomedical Engineering Research Organization, Japan** Graduate School of Science, The University of Tokyo, Japan***
○Mutsuko Kukimoto-Niino* Tetsuo Takagi* Ryogo Akasaka* Kazutaka Murayama** Tomomi Uchikubo-Kamo* Takaho Terada* Makoto Inoue* Satoru Watanabe* Akiko Tanaka* Yoshihide Hayashizaki* Takanori Kigawa* Mikako Shirouzu* Shigeyuki Yokoyama***
Rap2 interacting protein x (RPIPx) is a homolog of RPIP8, a specific effector of the Rap2 GTPase of the Ras family. The N-terminal region of RPIP8, which contains the RUN domain, interacts with Rap2. Using cell-free synthesis and NMR, we determined that the region encompassing residues 83-255 of mouse RPIPx, which is 40-residues larger than the predicted RUN domain (residues 113-245), is the minimum fragment that forms a correctly folded protein. This fragment, the RPIPx RUN domain, interacted specifically with Rap2B in vitro, in a nucleotide-dependent manner. The crystal structure of the RPIPx RUN domain was determined at 2.0 Å resolution, by the multiwavelength anomalous dispersion (MAD) method. The RPIPx RUN domain comprises eight anti-parallel a-helices, which form an extensive hydrophobic core, followed by an extended segment. This structure is distinct from any other known Ras/Rap-binding domain. The residues in the core region are highly conserved, suggesting the conservation of the RUN-domain fold among the RUN domain-containing proteins. The residues forming a positively charged surface are conserved between RPIP8 and its homologs, suggesting that this surface is important for Rap2 binding. In the crystal, the putative Rap2 binding site of the RPIPx RUN domain interacts with the extended segment in a segment-swapping manner.