STRUCTURE DETERMINATION OF THE RAS-BINDING DOMAIN OF THE RAL-SPECIFICGUANINE-NUCLEOTIDE EXCHANGE FACTOR RLF

Ral-specific guanine nucleotide exchange factors RalGDS, Rgl, and Rlf have been suggested to function as intermediates between Ras and Ral p athways by being able to bind Ras proteins through their C-terminal Ra s-binding domains (RBD). The RBDs of RalGDS and of the Ser/Thr kinase c-Raf-l have been shown to have the same tertiary structure. In contra st to the RBDs of Raf and RalGDS, which bind either Ras or Rap with hi gh affinity, Rlf-RBD has a similar affinity for both GTP-binding prote ins. To be able to compare these RBDs on a structural level, we have s olved the three-dimensional structure of Rlf-RBD by NMR spectroscopy. The overall tertiary structure of Rlf-RBD shows the beta beta alpha be ta beta alpha beta-fold of the ubiquitin superfamily and is very simil ar to that of RalGDS-RBD. The binding interface of:Rlf-RBD to Ras was mapped using chemical shift analysis and indicated a binding mode simi lar to that in the case of Rap.Raf-RBD. However, comparison of the put atively interacting regions revealed structural differences which are proposed to be responsible for the different substrate affinities of R lf-, RalGDS-, and Raf-RBD.