FIGURE 7.

Ionic interaction between Rap1 Glu⁴⁵ and KRIT1 Lys⁵⁷⁰ is important for high affinity binding. A, surface electrostatic potential of the Rap1 (left) and KRIT1 FERM (right) binding interface as open book view. The ionic interaction between Rap1 Glu⁴⁵ and KRIT1 Lys⁵⁷⁰ is highlighted. The dotted line separates the binding interface for KRIT1 F1 and F2 subdomains. B–E, binding of the KRIT1 FERM domain to Rap1 as analyzed on a Superdex-75 (10/300) GL gel filtration column at room temperature. B, incubation of KRIT1 WT with Rap1 WT results in complex formation. C, incubation of KRIT1 WT with Rap1(E45K) results in complex formation with a shift toward increased V_e compared with wild-type KRIT1, suggesting a reduced affinity. D, incubation of KRIT1(K570E) with Rap(E45K) results in complex formation with a shift comparable with wild-type complex, suggesting binding has been recovered. E, incubation of KRIT1(K570E) with Rap1 WT results in complex formation with a shift toward increased V_e compared with wild-type KRIT1, suggesting a reduced affinity. The shift is less significant than C.