2.5 Å structure of aspartate carbamoyltransferase complexed with the bisubstrate analog N-(phosphonacetyl)-l-aspartate

Krause, Kurt L.; Volz, Karl; Lipscomb, William N.

doi:10.1016/0022-2836(87)90265-8

Cited by 211 publications

(252 citation statements)

References 65 publications

Supporting

Mentioning

243

Contrasting

Order By: Relevance

“…In each catalytic site, residues Ser 80 and Lys 84 are contributed by one chain and the other residues are contributed by the adjacent chain (36). Since all these residues are conserved in the P. abyssi ATCase, its catalytic site should also be at the interface between two catalytic chains.…”

Section: Discussionmentioning

confidence: 99%

“…The presence of this cation ensures the proper folding of the domain for its interactions with the catalytic subunits. The catalytic sites are located at the interface between two catalytic chains belonging to the same trimer and involve residues belonging to both chains (36,44). These structural features were found in homologous enterobacterial enzymes.…”

mentioning

confidence: 96%

“…coli ATCase shows cooperativity for the binding of its substrate aspartate, a phenomenon which is explained by the transition from a T state having a low affinity for aspartate to an R state which has a high affinity for this substrate. The crystallographic structures of these two extreme conformations are known with a resolution of 2.5 Å (28,29,33,35,36). In the…”

mentioning

confidence: 99%

See 2 more Smart Citations

Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

et al. 1997

View full text Add to dashboard Cite

The genes coding for aspartate transcarbamylase (ATCase) in the deep-sea hyperthermophilic archaeon Pyrococcus abyssi were cloned by complementation of a pyrB Escherichia coli mutant. The sequence revealed the existence of a pyrBI operon, coding for a catalytic chain and a regulatory chain, as in Enterobacteriaceae.Comparison of primary sequences of the polypeptides encoded by the pyrB and pyrI genes with those of homologous eubacterial and eukaryotic chains showed a high degree of conservation of the residues which in E. coli ATCase are involved in catalysis and allosteric regulation. The regulatory chain shows more-extensive divergence with respect to that of E. coli and other Enterobacteriaceae than the catalytic chain. Several substitutions suggest the existence in P. abyssi ATCase of additional hydrophobic interactions and ionic bonds which are probably involved in protein stabilization at high temperatures. The catalytic chain presents a secondary structure similar to that of the E. coli enzyme. Modeling of the tridimensional structure of this chain provides a folding close to that of the E. coli protein in spite of several significant differences. Conservation of numerous pairs of residues involved in the interfaces between different chains or subunits in E. coli ATCase suggests that the P. abyssi enzyme has a quaternary structure similar to that of the E. coli enzyme. P. abyssi ATCase expressed in transgenic E. coli cells exhibited reduced cooperativity for aspartate binding and sensitivity to allosteric effectors, as well as a decreased thermostability and barostability, suggesting that in P. abyssi cells this enzyme is further stabilized through its association with other cellular components.Studies of extremophilic organisms provide information on the molecular mechanisms of biological adaptation to extreme environments. In this regard, Pyrococcus abyssi is of particular interest, being a hyperthermophilic and barophilic/barotolerant archaeon. This euryarchaebacterium, isolated from a deepsea hydrothermal vent located 2,000 m deep in the North Fiji Basin, was characterized by Erauso et al. (15,16). It is a heterotrophic and strictly anaerobic microorganism and belongs to the sulfur-metabolizing group. At atmospheric pressure, it grows at 67 to 102°C, with an optimum at 96°C. Hydrostatic pressure slightly increases the growth rate of P. abyssi as well as its optimal and maximal growth temperatures (16).Aspartate transcarbamylase (ATCase) (EC 2.1.3.2) is the first enzyme of the pyrimidine biosynthetic pathway. It catalyzes the carbamylation of the amino group of aspartate by carbamylphosphate with formation of carbamylaspartate and phosphate. ATCase is extensively studied as a model for structure-function relationships in cooperativity and allosteric regulation mechanisms, as well as for the evolution of these mechanisms from prokaryotes to humans. The structure and properties of the Escherichia coli enzyme have been studied in detail (for reviews, see references 3, 10, 26, 32, 40, and 70).This dodecame...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 96%

mentioning

confidence: 99%

See 1 more Smart Citation

Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

et al. 1997

View full text Add to dashboard Cite

show abstract

“…Because allosteric proteins are oligomeric proteins, conformational changes must be transduced from the ligand-binding sites to neighboring subunits (10). That such changes in quaternary structure accompany allosteric transitions has been shown at the atomic level by x-ray crystallography for many proteins-e.g., hemoglobin (11) and aspartate carbamoyltransferase (12,13). These examples show that the subunit interfaces play a pivotal role in the interconversion of different allosteric states, which are characterized by different quaternary structures.…”

mentioning

confidence: 99%

Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking

Watty

Methfessel

Hucho

1997

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

View full text Add to dashboard Cite

Receptor activity can be described in terms of ligand-induced transitions between functional states. The nicotinic acetylcholine receptor (nAChR), a prototypic ligand-gated ion channel, is an ''unconventional allosteric protein'' which exists in at least three interconvertible conformations, referred to as resting (low agonist affinity, closed channel), activated (open channel), and desensitized (high agonist affinity, closed channel). Here we show that 3,3 -dimethyl suberimidate (DMS) is an agonistic bifunctional cross-linking reagent, which irreversibly ''freezes'' the nAChR in a high agonist affinity͞closed-channel state. The monofunctional homologue methyl acetoimidate, which is also a weak cholinergic agonist, has no such irreversible effect. Glutardialdehyde, a cross-linker that is not a cholinergic effector, fixes the receptor in a low-affinity state in the absence of carbamoylcholine, but, like DMS, in a highaffinity state in its presence. Covalent cross-linking thus allows us to arrest the nAChR in defined conformational states.The nicotinic acetylcholine receptor (nAChR) is a heteropentameric transmembrane glycoprotein with the subunit stoichiometry ␣ 2 ␤␥␦. The five subunits are arranged around a central pore, which is permeable for cations in the receptor's activated state (1-3).The nAChR is an allosteric protein (4, 5) existing in different interconvertible states (4, 6). In the absence of an agonist the receptor stays in a resting or closed channel state. After binding of two agonist molecules in a positive cooperative manner, the receptor is activated and the channel opens. During longer agonist exposure the receptor desensitizes, which means that the receptor closes while acetylcholine (ACh) is still bound (7).Although the nature of allosteric transitions in oligomeric proteins is still under debate (8, 9) there is agreement that distinct allosteric states are characterized by distinct protein conformations. Because allosteric proteins are oligomeric proteins, conformational changes must be transduced from the ligand-binding sites to neighboring subunits (10). That such changes in quaternary structure accompany allosteric transitions has been shown at the atomic level by x-ray crystallography for many proteins-e.g., hemoglobin (11) and aspartate carbamoyltransferase (12, 13). These examples show that the subunit interfaces play a pivotal role in the interconversion of different allosteric states, which are characterized by different quaternary structures.For the nAChR it has been established that the two binding sites for agonists and competitive antagonists are each located at the interface between an ␣-subunit and the neighboring ␥-or ␦-subunit (14-18). Therefore one can assume that the contact sites between subunits play an important role in the function and allosteric properties for the nAChR as well.To date we know very little about the structural changes going on upon receptor activation and desensitization. Unwin and co-workers (19) examined the structure of the nAChR in the absenc...

show abstract

“…Binding of N-phosphonacetyl-L-aspartate (PALA, a transition-state analogue) results in the T to R transition, during which the allosteric-zinc interface opens 2-3 Å as the catalytic trimers separate and the catalytic and regulatory subunits rotate about their respective axes of symmetry (4)(5)(6)(7). Small-angle X-ray-scattering analysis indicated that there is no significant difference between the T state in the crystal and that in the solution forms, whereas the R state is more relaxed in solution than in the crystal.…”

mentioning

confidence: 99%

Allosteric Signal Transmission Involves Synergy between Discrete Structural Units of the Regulatory Subunit of Aspartate Transcarbamoylase

Liu

Wales

Wild

2000

Archives of Biochemistry and Biophysics

View full text Add to dashboard Cite

Previous studies have shown that the S5′ β-strand (r93-r97) of the regulatory polypeptides of the aspartate transcarbamoylases (ATCases) from Serratia marcescens and Escherichia coli are responsible for their diverged allosteric regulatory patterns, including conversion of CTP from an inhibitor in E. coli to an activator in S. marcescens. Similarly, mutation of residues located in the interface between the allosteric and the zinc domains resulted in conversion of the ATP responses of the E. coli enzyme from activation to inhibition, suggesting that this interface not only mediates but also discriminates the allosteric responses of ATP and CTP. To further decipher the roles and the interrelationships of these regions in allosteric communication, allosteric-zinc interface mutations (Y77F and V106A) have been introduced into both the native and the S5′ β-strand chimeric backgrounds. While the significance of this interface in the allosteric regulation has been confirmed, there is no direct evidence supporting the presence of distinct pathways for the ATP and CTP signals through this interface. The analysis of the mutational effects reported here suggested that the S5′ β-strand transmits the allosteric signal by modulating the hydrophobic allosteric-zinc interface rather than disturbing the allosteric ligand binding. Intragenic suppression by substitutions in the hydrophobic interface between the allosteric and the zinc domains of the regulatory chains resulted in the partial recovery of allosteric responses in the EC:rS5'sm chimera and reduced the activation by ATP in the Sm:rS5'ec chimera. Thus, it seems that there is a synergy between these two Structural Units. KeywordsATCase; allosterism; S5′ β-strand; allosteric-zinc interface; mutational effect; signal transmission; ligand binding Aspartate transcarbamoylase (EC 2.1.3.2; ATCase) 2 catalyzes the condensation of Laspartate with carbamoyl phosphate to form carbamoylaspartate and phosphate, ultimately leading to the pyrimidine end products, UTP and CTP (1-3). In Escherichia coli, the holoenzyme is composed of two catalytic subunits and three regulatory subunits (2c 3 :3r 2 ). Three catalytic polypeptides associate to form a functional catalytic subunit (trimer, c 3 ) and two regulatory polypeptides form a regulatory subunit (dimer, r 2 ) (Fig. 1) polypeptide is folded into an allosteric effector-binding domain (Allo domain, r1-r100) and a zinc-binding domain (Zn domain, r105-r146). The active sites are located at the interfaces of the Asp and CP domains of the catalytic trimers, positioned 60 Å away from the nucleotide-binding sites, which are located adjacent to the five-stranded β-sheets of the Allo domains and directly linked to the Zn domains through the S5′ β-strand. The hydrophobic allosteric-zinc interface includes leucine r32, phenylalanine r33, tyrosine r77, and valine r106. These residues are invariant among the four members of the Enterobacteriaceae whose sequences have been reported: E. coli, Serratia marcescens, Erwinia herbicola, and Salmone...

show abstract

2.5 Å structure of aspartate carbamoyltransferase complexed with the bisubstrate analog N-(phosphonacetyl)-l-aspartate

Cited by 211 publications

References 65 publications

Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking

Allosteric Signal Transmission Involves Synergy between Discrete Structural Units of the Regulatory Subunit of Aspartate Transcarbamoylase

Contact Info

Product

Resources

About