Affinity Labeling of the GDP/GTP Binding Site in Thermus Thermophilus Elongation Factor Tu

Summary

Affinity labeling of T. thermophilus EF-Tu•GDP(GTP) complexes by in situ oxidation with periodate results in specific modification of lysine residues 52, 137 and 325. Residue 52 of the native EF-Tu is modified by both GDP_OXI and GTP_OXI., but it cannot be modified by GDP_OXI if the protein is cleaved at arginine 59. Residue 137 is preferentially modified by GTP_OXI and is essentially unreactive toward GDP_OXI Cleavage of EF-Tu at position 59 renders an amino acid residue be very sensitive to reaction with GDP_OXI which is essentially unreactive in the native protein. Residue 325 is a minor reaction site accessible from both GDP_OXI and GTP_OXI in the native protein. EF-Tu nicked at position 59 does not show any reaction with GDP_OXI at lysine 325. Photoirradiation leads to crosslinking of the guanine moiety with region 181–190 of T. thermophilus EF-Tu. When the above affinity labeling results are fitted into the available three dimensional models of E. coli EF-Tu•GDP and H-ras protooncogen product p21•GDP complexes the following points emerge:

• 1) There is a high homology in the folding of the structural domains in both proteins.

• 2) The loop region connecting helix A with the β-sheet b (La Cour et al., 1985) of elongation factor Tu is placed in the vicinity of the bound GDP/GTP and in analogy to p21 it probably forms a part of a binding pocket for the nucleotide. This loop corresponds to the “effector loop” of G-proteins.

• 3) Cleavage at position 59 of EF-Tu leads to a conformational change resulting in altered reactivity of GDPOXI. towards lysine residues adjacent to the nucleotide binding pocket.

Dedicated to Professor Fritz Cramer on his 65 birthday.