Directed mutagenesis identifies amino acid residues involved in elongation factor Tu binding to yeast Phe-tRNAPhe

Abstract

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.

Figure 1

Four regions of EF-Tu interaction with yeast Phe-tRNA^Phe. (a) Schematic of T. aquaticus EF-Tu amino acids contacting yeast Phe-tRNA^Phe backbone. The hashed lines represent possible interactions between the amino acid and the tRNA up to 7Å. Region 1 contacts the 3′ end (green). Region 2 contacts the 5′ end (blue). Region 3 contacts the junction of the acceptor and T-stems (magenta). Region 4 contacts the T-stem (red). Residues which exhibit a ΔΔG⁰ >0.5 kcal/mol are highlighted in yellow.(b) Surface representation of yeast Phe-tRNA^Phe with regions contacted by EF-Tu highlighted. (c) Surface representation of T. aquaticus EF-Tu with yeast Phe-tRNA^Phe contact regions highlighted with the same color code as in (a).

Figure 2

Atomic details of the interactions of the four regions of T. aquaticus EF-Tu with the acceptor and T-stems of yeast Phe-tRNA^Phe from the co-crystal structure. The solid black lines represent possible contacting groups of EF-Tu and the tRNA within 7Å. (a) Region 1 interacting with the 3′ end of yeast Phe-tRNA^Phe. (b) Region 2 interacting with the 5′ end of yeast Phe-tRNA^Phe. (c) Region 3 interacting with the junction of the acceptor and T-stem of yeast Phe-tRNA^Phe. (d) Region 4 interacting with the T-stem of yeast Phe-tRNA^Phe.

Figure 3

Binding of T. thermophilus EF-Tu mutants to yeast Phe-tRNA^Phe in buffer B at 0⁰C. (a) Representative equilibrium binding curves fit to a single binding isotherm (●) wild type EF-Tu, K_D=15.6 nM, (◆) K52A, K_D= 34 nM, (○) N64A, K_D= 33.8 nM, (◇) H331V, K_D= 93.6 nM. (b) Representative active site titration curves with 300 nM of Phe-tRNA^Phe with the initial slopes for wild type and the same three mutations are fit to a linear equation and the intercept at 1.0 corresponds to 13, 6.3, 8.2, and 7.7 percent active EF-Tu, respectively. (c) Dissociation rate curves fit to single exponential for (●) wild type EF-Tu, k_off = 0.0016 s⁻¹, (◆) K52A, k_off = 0.0024 s⁻¹, (○) N64A, k_off = 0.0021s⁻¹ (◇) H331V, k_off = .0068 s⁻¹. (d) Dissociation rate curves of weak binding EF-Tu under tight binding conditions (50 mM NH₄Cl, 1 M NH₄SO₄) fit to single exponential for (●) wild-type EF-Tu k_off = .0002 s⁻¹, (▼) E271A, k_off = 0.019 s⁻¹, (□) R330A, k_off = 0.036 s⁻¹, (■) R389A, k_off =0.011 s⁻¹, (▲) No EF-Tu, rate = 0.35 s⁻¹.