Structure determination of the Ras-binding domain of the Ral-specific guanine 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 pathways by being able to bind Ras proteins through their C-terminal Ras-binding domains (RBD). The RBDs of RalGDS and of the Ser/Thr kinase c-Raf-1 have been shown to have the same tertiary structure. In contrast to the RBDs of Raf and RalGDS, which bind either Ras or Rap with high affinity, Rlf-RBD has a similar affinity for both GTP-binding proteins. To be able to compare these RBDs on a structural level, we have solved the three-dimensional structure of Rlf-RBD by NMR spectroscopy. The overall tertiary structure of Rlf-RBD shows the betabetaalphabetabetaalphabeta-fold of the ubiquitin superfamily and is very similar to that of RalGDS-RBD. The binding interface of Rlf-RBD to Ras was mapped using chemical shift analysis and indicated a binding mode similar to that in the case of Rap.Raf-RBD. However, comparison of the putatively interacting regions revealed structural differences which are proposed to be responsible for the different substrate affinities of Rlf-, RalGDS-, and Raf-RBD.
Esser
D
D
Max-Planck-Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, Abteilung Physikalische Biochemie, Dortmund, Germany.
Bauer
B
B
Wolthuis
R M
RM
Wittinghofer
A
A
Cool
R H
RH
Bayer
P
P
D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006165:Guanylyl Imidodiphosphate / Q000737:chemistry; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D020726:ral Guanine Nucleotide Exchange Factor; D020668:rap GTP-Binding Proteins; D018631:ras Proteins / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; C490805:Rgl2 protein, mouse; D014157:Transcription Factors; D020726:ral Guanine Nucleotide Exchange Factor; D006165:Guanylyl Imidodiphosphate; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D018631:ras Proteins