Monitoring the Transition from the T to the R State in E.coli Aspartate Transcarbamoylase by X-ray Crystallography: Crystal Structures of the E50A Mutant Enzyme in Four Distinct Allosteric States

Stieglitz, Kimberly A.; Stec, Boguslaw; Baker, Darren P.; Kantrowitz, Evan R.

doi:10.1016/j.jmb.2004.06.002

Cited by 35 publications

(39 citation statements)

References 40 publications

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“…All data sets were recorded at a static magnetic field of 18.8 T using a spectrometer equipped with a room-temperature probe-head. Shown also are the X-ray derived structures of each protein complex [12,60,61] and the dimensions of each molecule. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.…”

Section: A Simple Strategy For Preserving Magnetization Emerges From mentioning

confidence: 99%

Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view

Kay

2011

Journal of Magnetic Resonance

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Section: A Simple Strategy For Preserving Magnetization Emerges From mentioning

confidence: 99%

Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view

Kay

2011

Journal of Magnetic Resonance

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“…The initial model for the structure in the presence of CP was the T-state structure of the E50A catalytic chain mutant with phosphonoacetamide bound (PDB ID code 1TU0) (25). After initial rigid body, simulated annealing, minimization, and Bfactor refinement, inspection of initial maps and the value of the R factor indicated that model building was necessary.…”

Section: X-ray Data Collection and Processingmentioning

confidence: 99%

Structural basis for ordered substrate binding and cooperativity in aspartate transcarbamoylase

Wang

Stieglitz²,

Cardia³

et al. 2005

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

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X-ray structures of aspartate transcarbamoylase in the absence and presence of the first substrate carbamoyl phosphate are reported. These two structures in conjunction with in silico docking experiments provide snapshots of critical events in the function of the enzyme. The ordered substrate binding, observed experimentally, can now be structurally explained by a conformational change induced upon the binding of carbamoyl phosphate. This induced fit dramatically alters the electrostatics of the active site, creating a binding pocket for aspartate. Upon aspartate binding, a further change in electrostatics causes a second induced fit, the domain closure. This domain closure acts as a clamp that both facilitates catalysis by approximation and also initiates the global conformational change that manifests homotropic cooperativity.allosteric transition ͉ induced fit ͉ homotropic cooperativity E nzymes that exhibit positive cooperativity play a vital role in the regulation of the rates of key metabolic pathways by amplifying the response of a pathway to an effector molecule. In the case of aspartate transcarbamoylase (ATCase), substrateinduced domain closure triggers a quaternary conformational change that results in the observed homotropic cooperativity, one mechanism by which this enzyme controls the rate of de novo pyrimidine biosynthesis. Understanding the molecular features of domain closure not only provides insights into the mechanism of homotropic cooperativity but also demonstrates how ligandinduced domain closure can be used as part of catalytic mechanism of many enzymes (1, 2).The Escherichia coli ATCase is composed of six chains (M r 34,000 each) grouped into two trimeric catalytic subunits and six chains (M r 17,000 each) grouped into three dimeric regulatory subunits. The three active sites in the catalytic subunit are shared across the interface between adjacent chains (3, 4), whereas the regulatory subunits contain the binding sites for the heterotropic activator, ATP, as well as the heterotropic inhibitors, CTP and UTP. Each catalytic chain is composed of two structural domains, the carbamoyl phosphate (CP) domain (residues 1-135 and 292-310) and the L-aspartate (Asp) domain (residues 136-291), which contain the binding sites for CP and Asp, respectively. Each regulatory chain is also composed of two structural domains, the AL domain (residues 1-100) and the Zn domain (residues 101-153), which contain the binding sites for the allosteric effectors and the structural Zn, respectively. In mammals ATCase exists as a component of the multienzyme complex CAD (carbamoyl phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase) (5), and in humans, CAD has become a target for the development of antiproliferation drugs (6). The E. coli enzyme and the ATCase portion of CAD are 44% conserved, and all residues known to be involved in substrate binding and catalysis are conserved. Domain closure is triggered by the ordered binding of the substrates (7), with CP binding before Asp, and N-carbamoyl-L-...

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“…The planar angle is defined as the angle formed between the centers of gravity of the two domains and a hinge point. 20 The planar angles of the R PM , R PALA and R 236_PA structure (average of C1 and C6 chain) were 127.8°, 128.0°, 128.6° respectively. The increased angle between the CP and Asp domains results in an active site cavity that is more open in the R 236_PA structure than in the other Rstate structures.…”

Section: Resultsmentioning

confidence: 94%

Structural Model of the R State of Escherichia coli Aspartate Transcarbamoylase with Substrates Bound

Wang

Eldo

Kantrowitz

2007

Journal of Molecular Biology

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The allosteric enzyme aspartate transcarbamoylase (ATCase) exists in two conformational states. The enzyme, in the absence of substrates is primarily in the low-activity T state, is converted to the high-activity R state upon substrate binding, and remains in the R state until substrates are exhausted. These conformational changes have made it difficult to obtain structural data on R-state active-site complexes. Here we report the R-state structure of ATCase with the substrate Asp and the substrate analogue phosphonactamide (PAM) bound. This R-state structure represents the stage in the catalytic mechanism immediately before the formation of the covalent bond between the nitrogen of the amino group of Asp and the carbonyl carbon of carbamoyl phosphate. The binding mode of the PAM is similar to the binding mode of the phosphonate moiety of N-(phosphonoacetyl)-L-aspartate (PALA), the carboxylates of Asp interact with the same residues that interact with the carboxylates of PALA, although the position and orientations are shifted. The amino group of Asp is 2.9 Å away from the carbonyl oxygen of PAM, positioned correctly for the nucleophilic attack. Arg105 and Leu267 in the catalytic chain interact with PAM and Asp and help to position the substrates correctly for catalysis. This structure fills a key gap in the structural determination of each of the steps in the catalytic cycle. By combining these data with previously determined structures we can now visualize the allosteric transition through detailed atomic motions that underlie the molecular mechanism.

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Monitoring the Transition from the T to the R State in E.coli Aspartate Transcarbamoylase by X-ray Crystallography: Crystal Structures of the E50A Mutant Enzyme in Four Distinct Allosteric States

Cited by 35 publications

References 40 publications

Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view

Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view

Structural basis for ordered substrate binding and cooperativity in aspartate transcarbamoylase

Structural Model of the R State of Escherichia coli Aspartate Transcarbamoylase with Substrates Bound

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