Directed mutagenesis identifies amino acid residues involved in elongation factor Tu binding to yeast Phe-tRNAPhe.
The co-crystal structure of Thermus aquaticus elongation factor Tu.guanosine 5'- [beta,gamma-imido]triphosphate (EF-Tu.GDPNP) bound to yeast Phe-tRNA(Phe) reveals that EF-Tu interacts with the tRNA body primarily through contacts with the phosphodiester backbone. Twenty amino acids in the tRNA binding cleft of Thermus Thermophilus EF-Tu were each mutated to structurally conservative alternatives and the affinities of the mutant proteins to yeast Phe-tRNA(Phe) determined. Eleven of the 20 mutations reduced the binding affinity from fourfold to >100-fold, while the remaining ten had no effect. The thermodynamically important residues were spread over the entire tRNA binding interface, but were concentrated in the region which contacts the tRNA T-stem. Most of the data could be reconciled by considering the crystal structures of both free EF-Tu.GTP and the ternary complex and allowing for small (1.0 A) movements in the amino acid side-chains. Thus, despite the non-physiological crystallization conditions and crystal lattice interactions, the crystal structures reflect the biochemically relevant interaction in solution.
Sanderson
Lee E
LE
Northwestern University, Department of Biochemistry, Molecular Biology, and Cell Biology, 2205 Tech Drive Hogan 2-100. Evanston, IL, USA.
Uhlenbeck
Olke C
OC
D000596:Amino Acids / Q000235:genetics; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D012360:RNA, Transfer, Phe / Q000378:metabolism*; D016963:Thermus thermophilus / Q000235:genetics; D015003:Yeasts
D000596:Amino Acids; D050505:Mutant Proteins; D012360:RNA, Transfer, Phe; D010649:Phenylalanine; D010444:Peptide Elongation Factor Tu