Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase - PubMed (original) (raw)

Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase

Luc Fetler et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Many signaling and metabolic pathways rely on the ability of some of the proteins involved to undergo a substrate-induced transition between at least two structural states. Among the various models put forward to account for binding and activity curves of those allosteric proteins, the Monod, Wyman, and Changeux model for allostery theory has certainly been the most influential, although a central postulate, the preexisting equilibrium between the low-activity, low-affinity quaternary structure and the high-activity, high-affinity quaternary structure states in the absence of substrates, has long awaited direct experimental substantiation. Upon substrate binding, allosteric Escherichia coli aspartate transcarbamoylase adopts alternate quaternary structures, stabilized by a set of interdomain and intersubunit interactions, which are readily differentiated by their solution x-ray scattering curves. Disruption of a salt link, which is observed only in the low-activity, low-affinity quaternary structure, between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain yields a mutant enzyme that is in a reversible equilibrium between at least two states in the absence of ligand, a major tenet of the Monod, Wyman, and Changeux model. By using this mutant as a magnifying glass of the structural effect of ligand binding, a comparative analysis of the binding of carbamoyl phosphate (CP) and analogs points out the crucial role of the amine group of CP in facilitating the transition toward the high-activity, high-affinity quaternary state. Thus, the cooperative binding of aspartate in aspartate transcarbamoylase appears to result from the combination of the preexisting quaternary structure equilibrium with local changes induced by CP binding.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Solution x-ray scattering spectra of wild-type and D236A ATCase. Green, unliganded wild-type enzyme; black, unliganded D236A ATCase; red, wild-type enzyme in the presence of a 2-fold molar excess of PALA; orange, D236A ATCase in the presence of a 2-fold molar excess of PALA. (Inset) Solution x-ray scattering spectrum of the D236A ATCase with experimental error bars and linear combinations of wild-type enzyme extreme patterns. Black, unliganded D236A enzyme; magenta, 56% T-44% R; blue, 76% T-24% R.

Fig. 2.

Fig. 2.

Solution x-ray scattering spectra of D236A ATCase in the presence of nucleotide effectors. (A) pH 8.3. Black, unliganded D236A ATCase; brown, D236A ATCase + 5 mM ATP; blue, D236A ATCase + 5 mM CTP; green, unliganded wild-type ATCase (T-state). (B) pH 7. Black, unliganded D236A ATCase; green, D236A ATCase + 5 mM Mg-ATP; red, PALA-bound D236A ATCase + 5 mM Mg-ATP; blue, PALA-bound wild-type ATCase + 5 mM Mg-ATP.

Fig. 3.

Fig. 3.

Effect of temperature on both wild-type and D236A unliganded ATCases. (A) SAXS patterns of unliganded wild-type ATCase recorded at temperatures ranging from 4°C (dark blue) to 60°C (red). (B) SAXS patterns of unliganded D236A ATCase at temperatures ranging from 6°C (dark blue) to 45°C (red). (C) Reversibility of the temperature effect on the unliganded D236A ATCase. Scattering patterns recorded at 15°C (blue) and 20°C (orange) during the heating (continuous line) and cooling (dashed line) phases.

Fig. 4.

Fig. 4.

Solution x-ray scattering spectra of the D236A ATCase in the presence of the first substrate, CP, and its analogs. Black, unliganded D236A ATCase; blue, D236A ATCase + 5 mM CP; red, D236A ATCase + 2-fold molar excess of PALA; green, D236A ATCase + 5 mM _N_-methyl-carbamoyl phosphate; orange, D236A ATCase + 5 mM acetyl phosphate. (Inset) black, unliganded D236A ATCase; red, D236A ATCase + 100 mM aspartate.

Fig. 5.

Fig. 5.

Fitting of the unliganded D236A ATCase scattering pattern. Black, experimental SAXS pattern of the unliganded D236A ATCase with experimental error bars; red, linear combination of 62% T-38% Mg-ATP-bound D236A ATCase; green, linear combination of 63% T-37% Rsol+MgATP.

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