Site-directed mutagenesis of the GDP binding domain of bacterial elongation factor Tu.
The tertiary structure model of EF-Tu predicts that the amino acid sequence Val-Asp-His-Gly-Lys-Thr-Thr-Leu (residues 20-27) forms a pocket that binds the pyrophosphate group. To test this model we used site-directed mutagenesis to produce forms of EF-Tu altered in this region. The following mutations were constructed: Gly-20, Val-23, Glu-24, Ile-25, and Pro-27. Each protein was labeled with [35S]Met and was tested for its ability to interact with guanosine nucleotides and EF-Ts. The in vivo activity of each altered protein was tested by determining its ability to confer aurodox sensitivity to a resistant host. Mutations at residues 23, 24, 25, and 27 eliminated the ability of EF-Tu to interact with either guanosine nucleotides or EF-Ts in vitro, and these forms were also inactive in vivo. In contrast, the Gly-20 form was nearly as active as wild-type EF-Tu in vitro and in vivo. This mutation is theoretically equivalent to reversion of the Gly to Val transforming mutation of the cellular form of the ras gene product p21, a protein proposed to be structurally similar to EF-Tu in the GDP binding domain. In contrast to its effect in the ras gene, the Val to Gly conversion did not affect the endogenous GTPase of EF-Tu. We conclude that the tertiary structure model is correct in its assignment of the pyrophosphate binding site to residues 23-27; however, there are likely to be some significant differences between the configurations of the GTPases of EF-Tu and p21.
Hwang
Y W
YW
New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314.
McCabe
P G
PG
Innis
M A
MA
Miller
D L
DL
D000595:Amino Acid Sequence; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D012360:RNA, Transfer, Phe / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012685:Sepharose; D013329:Structure-Activity Relationship
D006150:Guanine Nucleotides; D012360:RNA, Transfer, Phe; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D012685:Sepharose; D019204:GTP-Binding Proteins; D010444:Peptide Elongation Factor Tu