The Role of Intersubunit Interactions for the Stabilization of the T State of Escherichia coli Aspartate Transcarbamoylase

Chan, Robin S.; Sakash, Jessica B.; Macol, Christine P.; West, Jay M.; Tsuruta, Hiro; Kantrowitz, Evan R.

doi:10.1074/jbc.m208919200

Cited by 10 publications

(11 citation statements)

References 39 publications

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“…As already reported (22), the scattering curve of the unliganded D236A ATCase cannot be approximated by a linear combination of the wild-type T and R curves. Indeed, a 56% T-44% R linear combination of the wild-type T and R patterns fits well the region of the first minimum and maximum but fails in the [0.022 Å Ϫ1 to 0.03 Å Ϫ1 ] region (the root-mean-square value of reduced residuals in this range is 3.3), which requires a 76% T-24% R combination [ Fig.…”

Section: Resultsmentioning

confidence: 96%

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

Fetler

Kantrowitz²,

Vachette³

2007

Proc. Natl. Acad. Sci. U.S.A.

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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 lowactivity, 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, lowaffinity 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.enzyme regulation ͉ homotropic cooperativity ͉ induced fit ͉ intersubunit interactions ͉ small-angle x-ray scattering M any cellular responses, such as cell signaling, cell movements, intermediary metabolism, and the selective expression of genes, rely on the capacity of proteins to switch between different stable conformations in a reversible manner (1). Crystallographic studies have shown that, in agreement with the Monod, Wyman, and Changeux model for allostery theory (MWC) (2), most such proteins are made up of a finite number of identical subunits regularly organized around symmetry axes, which increases the stability of their conformational states and enhances the abrupt, switch-like character of their quaternarystructure transition between a low-activity, low-affinity (T) state and a high-activity, high-affinity (R) state (3). A critical statement of the MWC was that this conformational transition involves states that are populated in the absence of ligand and may spontaneously interconvert, the ligand stabilizing the conformation to which it binds with higher affinity. Initially, these concepts were developed based on catalytic activity measurements performed with regulatory enzymes, such as aspartate transcarbamoylase from Escherichia coli (ATCase). Most of these data...

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Section: Resultsmentioning

confidence: 96%

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

Fetler

Kantrowitz²,

Vachette³

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…A particularly critical salt link is between Asp-236c1 and Lys-143r4. When this interaction is broken by mutation of either Lys-143r (52) or Asp-236c (53,54) the enzyme no longer exists in the T state. This interaction is lost in the K244N mutant (Fig.…”

Section: Structural Alterations Due To Mutations At 244 In Solution-mentioning

confidence: 99%

240s Loop Interactions Stabilize the T State of Escherichia coli Aspartate Transcarbamoylase

Alam

Stieglitz

Caban

et al. 2004

Journal of Biological Chemistry

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Here the functional and structural importance of interactions involving the 240s loop of the catalytic chain for the stabilization of the T state of aspartate transcarbamoylase were tested by replacement of Lys-244 with Asn and Ala. For the K244A and K244N mutant enzymes, the aspartate concentration required to achieve halfmaximal specific activity was reduced to 8.4 and 4.0 mM, respectively, as compared with 12.4 mM for the wild-type enzyme. Both mutant enzymes exhibited dramatic reductions in homotropic cooperativity and the ability of the heterotropic effectors to modulate activity. Small angle x-ray scattering studies showed that the unligated structure of the mutant enzymes, and the structure of the mutant enzymes ligated with N-phosphonacetyl-L-aspartate, were similar to that observed for the unligated and N-phosphonacetyl-L-aspartateligated wild-type enzyme. A saturating concentration of carbamoyl phosphate alone has little influence on the small angle x-ray scattering of the wild-type enzyme. However, carbamoyl phosphate was able to shift the structure of the two mutant enzymes dramatically toward R, establishing that the mutations had destabilized the T state of the enzyme. The x-ray crystal structure of K244N enzyme showed that numerous local T state stabilizing interactions involving 240s loop residues were lost. Furthermore, the structure established that the mutation induced additional alterations at the subunit interfaces, the active site, the relative position of the domains of the catalytic chains, and the allosteric domain of the regulatory chains. Most of these changes reflect motions toward the R state structure. However, the K244N mutation alone only changes local conformations of the enzyme to an R-like structure, without triggering the quaternary structural transition. These results suggest that loss of cooperativity and reduction in heterotropic effects is due to the dramatic destabilization of the T state of the enzyme by this mutation in the 240s loop of the catalytic chain.Regulation of metabolic pathways via enzymes whose activity is dependent upon the concentration of various metabolites is of paramount importance for all living systems. As a response to a change in the concentration of certain metabolites, a regulatory enzyme can alter its catalytic activity thereby regulating the flux of metabolic intermediates. In particular, a change in function (such as the affinity for substrate) necessarily mandates a change in structure (1). One excellent example of a regulatory enzyme in which there are dramatic functional and structural changes dependent upon the concentrations of substrates and allosteric effectors is Escherichia coli aspartate transcarbamoylase.Aspartate transcarbamoylase (EC 2.1.3.2) catalyzes the first reaction in pyrimidine biosynthesis, the reaction between carbamoyl phosphate and L-aspartate to form N-carbamoyl-L-aspartate and inorganic phosphate. The enzyme shows homotropic cooperativity for the substrate L-aspartate (2). Its activity is inhibited heterotropically by...

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“…Samples of 0.5 mM R164N Runt domain-DNA complex were prepared and spectra recorded at 40°C as described previously 24; 30; 37 . 15 The S-switch region, βE'-F loop, and sidechains of the Runt domain residues involved in interactions with DNA are shown. The P values for the differences in K 2 and K 4 were = 0.11 and 0.06, respectively.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

“…In order to assess whether this change in the S-switch dynamics is transmitted to other portions of the protein, we collected T 1 , T 2 , and heteronuclear NOE data on a [U-2 H, 15 N]-labeled (>98% 2 H) sample of the R164N Runt domain (complexed to DNA) and calculated the R 1 * R 2 data. As seen in Figure 5B, there is a clear reduction in the number of residues displaying exchange, including residues in the DNA-contacting βA'-B (A88) and βE'-F (G41) loops, and in the βF strand (T147, I150).…”

Section: The R164n Mutation Quenches Exchange In the Dynamic Regions mentioning

confidence: 99%

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A Mutation in the S-switch Region of the Runt Domain Alters the Dynamics of an Allosteric Network Responsible for CBFβ Regulation

Lukasik

Liu

et al. 2006

Journal of Molecular Biology

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SUMMARYThe Runt domain is the DNA binding domain of the core binding factor (CBF) Runx subunits. The CBFs are transcription factors that play critical roles in hematopoiesis, bone, and neuron development in mammals. A common non-DNA binding CBFβ subunit heterodimerizes with the Runt domain of the Runx proteins and allosterically regulates its affinity for DNA. Previous NMR dynamics studies suggested a model whereby CBFβ allosterically regulates DNA binding by quenching conformational exchange in the Runt domain, particularly in the S-switch region and the βE'-F loop. We sought to test this model, and to this end introduced all possible single amino acid substitutions into the S-switch region and the βE'-F loop, and screened for mutations that enhanced DNA-binding. We demonstrate that one Runt domain mutant, R164N, binds both DNA and CBFβ with higher affinity, but it is less sensitive to allosteric regulation by CBFβ. Analysis of NMR relaxation data shows that the chemical exchange exhibited by the wild-type Runt domain is largely quenched by the R164N substitution. These data support a model in which the dynamic behavior of a network of residues connecting the CBFβ and DNA binding sites on the Runt domain plays a critical role in the mechanism of allosteric regulation. This study provides an important functional link between dynamic behavior and protein allosteric function, consistent with results on other allosterically regulated proteins.

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The Role of Intersubunit Interactions for the Stabilization of the T State of Escherichia coli Aspartate Transcarbamoylase

Cited by 10 publications

References 39 publications

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

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

240s Loop Interactions Stabilize the T State of Escherichia coli Aspartate Transcarbamoylase

A Mutation in the S-switch Region of the Runt Domain Alters the Dynamics of an Allosteric Network Responsible for CBFβ Regulation

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