Structural consequences of mono-glucosylation of Ha-Ras by Clostridium sordellii lethal toxin

Mono-glucosylation of Ha-Ras by Clostridium sordellii lethal toxin at effector region threonine 35 has diverse effects on the Ras GTPase cycle, the dominant one of which is the inhibition of Ras-Raf coupling, leading to complete blockade of Ras downstream signaling. To understand the structural basis of the functional consequences of glucosylation, the X-ray crystal structure of glucosylated Ras-GDP was compared with that of non-modified Ras. Glucosylated Ras exhibits a different crystal packing but the overall three-dimensional structure is not altered. The glucose group does not affect the conformation of the effector loop. Due to steric constraints, the glucose moiety prevents the formation of the GTP conformation of the effector loop, which is a prerequisite for binding to the Raf-kinase. The X-ray crystal data also revealed the alpha-anomeric configuration of the bound glucose, indicating that the glucose transfer proceeds under retention of the C-1 configuration of the d-alpha-glucose. Therefore, glucosylation preserves the inactive conformation of the effector loop independently of the nucleotide occupancy, leading to a complete inhibition of downstream signaling of Ras.