TY  - GEN
T1  - Genetic analysis of the interaction between the N- and C-terminal halves of UNC-112 (kindlin)
AU  - Qadota, Hiroshi
AU  - Luo, Yating
AU  - Oberhauser, Andres F
AU  - Benian, Guy M
DO  - 10.17912/micropub.biology.000342
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000342/
AB  - C. elegans UNC-112 (kindlin) is required for muscle sarcomere assembly (Rogalski et al. 2000), and is part of a conserved four protein complex that associates with the cytoplasmic tail of integrin at the base of muscle integrin adhesion complexes (Mackinnon et al. 2002; Lin et al. 2003; Norman et al. 2007; Qadota et al. 2014). UNC-112 consists of 720 amino acids and contains FERM and PH domains. UNC-112 N-terminal half (1-396 aa) can bind to UNC-112 C-terminal half (397-720 aa), and this interaction is inhibited by the association of PAT-4 (integrin linked kinase, ILK) to the UNC-112 N-terminal half (Qadota et al. 2012). The UNC-112 N-terminal half with either T346A or E349K mutations cannot bind to the UNC-112 C-terminal half, but can still bind to PAT-4 (ILK) (Qadota et al. 2012). To elucidate the molecular mechanism by which the UNC-112 N- and C-terminal halves interact genetically, we identified suppressor mutations in the UNC-112 C-terminal half that restore the ability of the UNC-112 C-terminal half to bind to the UNC-112 N-terminal half with T346A or E349K. We introduced random mutations into the UNC-112 C-terminal half (by error-prone PCR), and assayed for binding to the UNC-112 N-terminal half with T346A or E349K. From screening using the yeast two hybrid system and DNA sequencing of suppressor clones, we identified 5 single amino acid changes; R633G, S644C, N659D, I662T, and R663Q. S644C and R663Q were identified from screening with the UNC-112 N-terminal half containing T346A, and R633G, N659D, and I662T were identified from screening with the UNC-112 N-terminal half containing E349K. The wild type and all 5 suppressor clones failed to activate growth with an empty prey vector. Our results are summarized in Figure 1A. The UNC-112 C-terminal half with S644C or R663Q can bind to UNC-112 N-terminal half with T346A as expected from screening (see Methods), but also can bind to the UNC-112 N-terminal half with E349K. The UNC-112 C-terminal half with R633G, N659D, or I662T can bind to the UNC-112 N-terminal half with E349K as expected and but can also bind to UNC-112 N-terminal half with T346A. Interestingly, these 5 single amino acid changes are located within a 30 amino acid long segment (633 to 663; yellow bar in Figure 1). We speculate that these 30 amino acid residues of UNC-112 C-terminal half form a “Pocket” corresponding to the T346-E349 region of UNC-112 N-terminal half as a “Key”. We have shown previously, that only full-length UNC-112 can bind to cytoplasmic tail of PAT-3 (beta-integrin) by yeast two hybrid and biochemical methods (Qadota et al. 2012). Full length UNC-112 singly harboring the 5 suppressor mutations were tested for binding to the cytoplasmic tail of PAT-3. We found that 4 of 5 suppressors (R633G, S644C, I662T, and R663Q) could still bind to PAT-3, but full length UNC-112 with N659D could not bind (Figure 1B), suggesting that N659D causes a severe conformational change.
PY  - 2020
JO  - microPublication Biology
ER  -