A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase

Abstract

The 306-kDa aspartate transcarbamoylase is a well studied regulatory enzyme, and it has emerged as a paradigm for understanding allostery and cooperative binding processes. Although there is a consensus that the cooperative binding of active site ligands follows the Monod-Wyman-Changeux (MWC) model of allostery, there is some debate about the binding of effectors such as ATP and CTP and how they influence the allosteric equilibrium between R and T states of the enzyme. In this article, the binding of substrates, substrate analogues, and nucleotides is studied, along with their effect on the R-T equilibrium by using highly deuterated, 1 H, 13 C-methyl-labeled protein in concert with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR. Although only the T state of the enzyme can be observed in spectra of wild-type unliganded aspartate transcarbamoylase, binding of active-site substrates shift the equilibrium so that correlations from the R state become visible, allowing the equilibrium constant (L ) between ligand-saturated R and T forms of the enzyme to be measured quantitatively. The equilibrium constant between unliganded R and T forms (L) also is obtained, despite the fact that the R state is ''invisible'' in spectra, by means of an indirect process that makes use of relations that emerge from the fact that ligand binding and the R-T equilibrium are linked. Titrations with MgATP unequivocally establish that its binding directly perturbs the R-T equilibrium, consistent with the Monod-Wyman-Changeux model. This study emphasizes the utility of modern solution NMR spectroscopy in understanding protein function, even for systems with aggregate molecular masses in the hundreds of kilodaltons.allostery ͉ Monod-Wyman-Changeux (MWC) model ͉ NMR spectroscopy ͉ methyl-transverse relaxation optimized spectroscopy ͉ ligand binding Q uantitative, site-specific studies of proteins by NMR spectroscopy for the most part have been restricted to systems with molecular masses on the order of 50 kDa or less. With the development of new labeling schemes along with experiments that optimally preserve NMR signals, it now has become possible to investigate much larger complexes (1). For example, Wüth-rich, Horwich, and coworkers have used 1 H-15 N cross-correlated relaxation-induced polarization transfer (CRIPT) spectroscopy to establish which residues of highly deuterated GroES interact with GroEL in a GroES-GroEL complex that is 900 kDa (2) and to study interactions between substrates and GroEL (3). Sprangers and coworkers have exploited methyl-transverse relaxation optimized spectroscopy (TROSY) of 1 H, 13 C-methyl-labeled probes in the 300-kDa highly deuterated protease ClpP to quantify dynamics and relate it to function (4). In a second study, this methyl-based approach has been used to measure sitespecific dynamics in the 20S proteasome (670 kDa) along with binding to target molecules (5).The methodology that now is available opens the possibility for the study of a wide range of molecular machines in a site-specific...

Trapping and structure determination of an intermediate in the allosteric transition of aspartate transcarbamoylase

“…These results suggest that the previously reported findings that the K164E/E239K ATCase is in the R state may need to be reevaluated (2,3). The evidence presented here clearly demonstrates that the quaternary structure of the K164E/E239K ATCase is not identical to the wild-type ATCase-PALA complex, the typical structure used to define the R state.…”

“…The solution NMR report by Velyvis et al (3) also asserts the K164E/E239K ATCase is in the R state and concludes that the Monod, Wyman, and Changeux model (13) can fully account for the allosteric properties of ATCase. The experiments performed by Velyvis et al (3) were carried out at 37°C and pH 7.5. At this pH value and 25°C, the K164E/E239K ATCase is not in the canonical R-state structure (Fig.…”

“…The enzymatic properties of the double mutant (K164E/E239K ATCase) include a lack of homotropic cooperativity and an inability to be activated by ATP or inhibited by CTP: properties coincident of an enzyme that cannot transition between the two allosteric states (2). More recently, Velyvis et al (3) demonstrated that the partially labeled K164E/E239K ATCase exhibited certain chemical shifts in solution NMR studies that they conclude are characteristic of the R state of the enzyme. These same chemical shifts were also observed when the wild-type enzyme was bound with the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) (3), which is known to stabilize fully the R state of the enzyme (4).…”

Dissecting Enzyme Regulation by Multiple Allosteric Effectors: Nucleotide Regulation of Aspartate Transcarbamoylase

“…Quantitative modeling of the enzyme activity as a function of ATP and CTP binding has been performed and generally agrees with the classical model of Monod et al (19), in which the enzyme complex exists in either a T (tense and less active) or R (relaxed and more active) state, with the transition between the two states sensitive to ATP, CTP, and substrate concentrations (12,(20)(21)(22).…”

NMR reveals novel mechanisms of protein activity regulation

“…Thanks to the pioneering work from the lab of Lewis Kay it is now possible to obtain both structural and dynamical information on supramolecular protein machineries. [93][94][95][96][97] The key to achieving this has been the development of a labeling protocol that allows the specific protonation of methyl groups in an otherwise deuterated background. 94,98,99 The approach exploits some very favorable properties of methyl groups in proteins: (i) they occur frequently in the hydrophobic cores of proteins, or at the interfaces of biomolecular complexes, and are thus excellent reporters of structure and dynamics; (ii) the three protons of the methyl group all contribute to the intensity of the same signal, and therefore methyl probes are significantly more sensitive than other candidates.…”