Guanine nucleotide exchange factor independence of the G-protein eEF1A through novel mutant forms and biochemical properties

Abstract

Most G-proteins require a guanine nucleotide exchange factor (GEF) to regulate a variety of critical cellular processes. Interestingly, a small number of G-proteins switch between the active and inactive forms without a GEF. Translation elongation factor 1A (eEF1A) normally requires the GEF eEF1Balpha to accelerate nucleotide dissociation. However, several mutant forms of eEF1A are functional independent of this essential regulator in vivo. GEF-independent eEF1A mutations localize close to the G-protein motifs that are crucial for nucleotide binding. Kinetic analysis demonstrated that reduced GDP affinity correlates with wild type growth and high translation activities of GEF-independent mutants. Furthermore, the mutant forms show an 11-22-fold increase in rates of GDP dissociation from eEF1A compared with the wild type protein. All mutant forms have dramatically enhanced stability at elevated temperatures. This, coupled with data demonstrating that eEF1A is also more stable in the presence of nucleotides, suggests that both the GEF and nucleotide have stabilizing effects on eEF1A. The biochemical properties of these eEF1A mutants provide insight into the mechanism behind GEF-independent G-protein function.

FIGURE 1.

GEF-independent mutants of eEF1A reduce GDP binding. Equilibrium dissociation constants (K_d) for the mutant forms of eEF1A and mant-GDP were measured. Aliquots of mant-GDP were added to binding buffer without (A) or with 1 μm wild type (B) or the mutant forms (C–E) of eEF1A. The fluorescence was measured by FRET via excitation at 280 nm and emission of 440 nm for mant moiety. Fluorescence intensity (cpm) versus the concentration of mant-GDP (μm) was plotted, and data (•) were fit to a hyperbolic curve to obtain the K_d value. The K_d values are measured as 3.54 (T22S) (C), 2.66 (R164K) (D), 1.80 (A112T) (E), and 1.18 μm (A117V) (F). Residuals for the fits are shown in the lower panels to detect the experimental error for the fitted data sets.

FIGURE 2.

GEF-independent mutants of eEF1A do not affect GMPPNP binding. The K_d values for the wild type and mutant forms of eEF1A and mant-GMPPNP were measured. Aliquots of mant-GMPPNP were added to binding buffer without (A) or with 1 μm of wild type (B) or mutant forms (C–E) of eEF1A. The fluorescence was measured and plotted as in Fig. 1. The K_d values are measured as 0.47 (T22S) (C), 0.41 (R164K) (D), 1.01 (A112T) (E), and 1.09 μm (A117V) (F). Residuals for the fits are shown in the lower panels to detect the experimental error for the fitted data sets.

FIGURE 3.

Mutant forms of eEF1A demonstrate faster GDP dissociation. GDP dissociation rate constants (k) for the wild type and the mutant forms of eEF1A were measured. Mutant forms of eEF1A (A–D) prebound to mant-GDP were rapidly mixed with excess GDP. The rate of mant-nucleotide release was monitored as a decrease in fluorescent intensity over time and fitted by a single-exponential decay equation to obtain k_off values. The k_off values are measured as 3.89 (A117V) (A), 2.54 (R164K) (B), 1.96 (A112T) (C), and 1.88 s^-1 (T22S) (D). Residuals for the fits are shown in the lower panels to detect the experimental error for the fitted data sets.

FIGURE 4.

Mutant forms of eEF1A show enhanced thermostability compared with the wild type protein. The wavelength spectrums of the 0.2–0.4 mg/ml purified proteins are carried out at the indicated temperatures. A, circular dichroism spectra of wild type eEF1A (• and ○) and A112T (▴ and ▵) forms of eEF1A. The closed symbols represent the wavelength spectrum at the native state (25 °C), whereas open symbols represent the spectrum at 70 °C. B, circular dichroism spectra of the mutant forms of eEF1A, R146K (○), T22S (▴), A112T (▵), and A117V (▪), in comparison with wild type (•) at 70°C.

FIGURE 5.

GEF-independent mutants increase eEF1A stability. Unfolding intermediates of the wild type (A), A112T (B), R164K (C), A117V (D), and T22S (E) forms of eEF1A measured by circular dichroism spectra and collected as a function of temperature (left). The spectra were deconvoluted into three basis curves using the convex constraint algorithm. Each curve (▪, ▵, and •) represents a different state of unfolding. The right panel shows the fraction of each basis curve contributing to each spectrum at each temperature.

FIGURE 6.

Guanine nucleotides increase eEF1A stability. Circular dichroism spectra of apo-eEF1A (A), eEF1A with GDP (B), and eEF1A with GMPPNP (C) were measured. The spectra were deconvoluted into three basis curves using the convex constraint algorithm. Each curve (▪, ▵, and •) represents a different state of unfolding.

FIGURE 7.

GEF-independent mutants of eEF1A either affect the P-loop or the NKXD element. A, Thr-22 (magenta) and Ala-112 (cyan) residues and their positions respective to the nucleotide (green) and the P-loop (magenta) are shown. B, Arg-164 (wheat) and Ala-117 (purple) residues and their positions respective to the nucleotide (green) and the NKXD motif (yellow) are shown. The structure was produced with the PyMOL program (32), using the co-crystal structure of the eEF1A·eEF1Bα complex with GDP (Protein Data Bank code 1IJE) (39). C, sequence alignment of yeast eEF1A with the G-proteins independent of GEF in yeast (eEF2, eIF5B, eRF3, Guf1p, and Hbs1p) and human (eEF2, eIF5B, eRF3, Guf1p, Hbs1-like eRFs, and eEFSec). The alignment was generated by Jalview (41).