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Crystal structures of phosphonoacetamide ligated T and phosphonoacetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8-A resolution and neutral pH.

Gouaux JE, Lipscomb WN
Biochemistry 29 389-402 (1990)
Related entries: 2at1, 3at1

Cited: 40 times
EuropePMC logo PMID: 2405902

Abstract

The T----R transition of the cooperative enzyme aspartate carbamoyltransferase occurs at pH 7 in single crystals without visibly cracking many of the crystals and leaving those uncracked suitable for single-crystal X-ray analysis. To promote the T----R transition, we employ the competitive inhibitors of carbamoyl phosphate and aspartate, which are phosphonoacetamide (PAM) and malonate, respectively. In response to PAM binding to the T-state crystals, residues Thr 53-Thr 55 and Pro 266-Pro 268 move to their R-state positions to bind to the phosphonate and amino group of PAM. These changes induce a conformation that can bind tightly the aspartate analogue malonate, which thereby effects the allosteric transition. We prove this by showing that PAM-ligated T-state crystals (Tpam), space group P321 (a = 122.2 A, c = 142.2 A), when transferred to a solution containing 20 mM PAM and 8 mM malonate at pH 7, isomerize to R-state crystals (Rpam,mal,soak), space group also P321 (a = 122.2 A, c = 156.4 A). The R-state structure in which the T----R transition occurs within the crystal at pH 7 compares very well (rms = 0.19 A for all atoms) with an R-state structure determined at pH 7 in which the crystals were initially grown in a solution of PAM and malonate at pH 5.9 and subsequently transferred to a buffer containing the ligands at pH 7 (Rpam,mal,crys). In fact, both of the PAM and malonate ligated R-state structures are very similar to both the carbamoyl phosphate and succinate or the N-(phosphonoacetyl)-L-aspartate ligated structures, even though the R-state structures reported here were determined at pH 7. Crystallographic residuals refined to 0.16-0.18 at 2.8-A resolution for the three structures.

Reviews - 1at1 mentioned but not cited (1)

  1. Allostery and cooperativity in Escherichia coli aspartate transcarbamoylase. Kantrowitz ER. Arch Biochem Biophys 519 81-90 (2012)

Articles - 1at1 mentioned but not cited (4)

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Reviews citing this publication (4)

  1. Structural symmetry and protein function. Goodsell DS, Olson AJ. Annu Rev Biophys Biomol Struct 29 105-153 (2000)
  2. Structure and mechanisms of Escherichia coli aspartate transcarbamoylase. Lipscomb WN, Kantrowitz ER. Acc Chem Res 45 444-453 (2012)
  3. From Genome to Structure and Back Again: A Family Portrait of the Transcarbamylases. Shi D, Allewell NM, Tuchman M. Int J Mol Sci 16 18836-18864 (2015)
  4. Modelling allosteric processes in E coli aspartate transcarbamylase. Cherfils J, Vachette P, Janin J. Biochimie 72 617-624 (1990)

Articles citing this publication (31)

  1. Using a library of structural templates to recognise catalytic sites and explore their evolution in homologous families. Torrance JW, Bartlett GJ, Porter CT, Thornton JM. J Mol Biol 347 565-581 (2005)
  2. Insights into the mechanisms of catalysis and heterotropic regulation of Escherichia coli aspartate transcarbamoylase based upon a structure of the enzyme complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate at 2.1 A. Jin L, Stec B, Lipscomb WN, Kantrowitz ER. Proteins 37 729-742 (1999)
  3. Human ornithine transcarbamylase: crystallographic insights into substrate recognition and conformational changes. Shi D, Morizono H, Yu X, Tong L, Allewell NM, Tuchman M. Biochem J 354 501-509 (2001)
  4. Crystal structure of CTP-ligated T state aspartate transcarbamoylase at 2.5 A resolution: implications for ATCase mutants and the mechanism of negative cooperativity. Kosman RP, Gouaux JE, Lipscomb WN. Proteins 15 147-176 (1993)
  5. Large differences are observed between the crystal and solution quaternary structures of allosteric aspartate transcarbamylase in the R state. Svergun DI, Barberato C, Koch MH, Fetler L, Vachette P. Proteins 27 110-117 (1997)
  6. Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase. Fetler L, Kantrowitz ER, Vachette P. Proc Natl Acad Sci U S A 104 495-500 (2007)
  7. A molecular mechanism for pyrimidine and purine nucleotide control of aspartate transcarbamoylase. Stevens RC, Lipscomb WN. Proc Natl Acad Sci U S A 89 5281-5285 (1992)
  8. Crystal structure of human ornithine transcarbamylase complexed with carbamoyl phosphate and L-norvaline at 1.9 A resolution. Shi D, Morizono H, Aoyagi M, Tuchman M, Allewell NM. Proteins 39 271-277 (2000)
  9. 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 K, Stec B, Baker DP, Kantrowitz ER. J Mol Biol 341 853-868 (2004)
  10. Application of methyl-TROSY NMR to test allosteric models describing effects of nucleotide binding to aspartate transcarbamoylase. Velyvis A, Schachman HK, Kay LE. J Mol Biol 387 540-547 (2009)
  11. Heterotropic interactions in Escherichia coli aspartate transcarbamylase. Subunit interfaces involved in CTP inhibition and ATP activation. Xi XG, van Vliet F, Ladjimi MM, de Wannemaeker B, de Staercke C, Glansdorff N, Piérard A, Cunin R, Hervé G. J Mol Biol 220 789-799 (1991)
  12. The regulatory subunit of Escherichia coli aspartate carbamoyltransferase may influence homotropic cooperativity and heterotropic interactions by a direct interaction with the loop containing residues 230-245 of the catalytic chain. Newton CJ, Kantrowitz ER. Proc Natl Acad Sci U S A 87 2309-2313 (1990)
  13. Arginine 54 in the active site of Escherichia coli aspartate transcarbamoylase is critical for catalysis: a site-specific mutagenesis, NMR, and X-ray crystallographic study. Stebbins JW, Robertson DE, Roberts MF, Stevens RC, Lipscomb WN, Kantrowitz ER. Protein Sci 1 1435-1446 (1992)
  14. Apparent cooperativity for carbamoylphosphate in Escherichia coli aspartate transcarbamoylase only reflects cooperativity for aspartate. England P, Leconte C, Tauc P, Hervé G. Eur J Biochem 222 775-780 (1994)
  15. A single mutation in the regulatory chain of Escherichia coli aspartate transcarbamoylase results in an extreme T-state structure. Williams MK, Stec B, Kantrowitz ER. J Mol Biol 281 121-134 (1998)
  16. Molecular dynamics simulations and rigid body (TLS) analysis of aspartate carbamoyltransferase: evidence for an uncoupled R state. Tanner JJ, Smith PE, Krause KL. Protein Sci 2 927-935 (1993)
  17. Aspartate carbamoyltransferase from the thermoacidophilic archaeon Sulfolobus acidocaldarius. Cloning, sequence analysis, enzyme purification and characterization. Durbecq V, Thia-Toong TL, Charlier D, Villeret V, Roovers M, Wattiez R, Legrain C, Glansdorff N. Eur J Biochem 264 233-241 (1999)
  18. Conversion of the allosteric regulatory patterns of aspartate transcarbamoylase by exchange of a single beta-strand between diverged regulatory chains. Liu L, Wales ME, Wild JR. Biochemistry 36 3126-3132 (1997)
  19. Cooperativity in Bacillus stearothermophilus pyruvate kinase. Lovell SC, Mullick AH, Muirhead H. J Mol Biol 276 839-851 (1998)
  20. Allosteric control of quaternary states in E. coli aspartate transcarbamylase. Stevens RC, Lipscomb WN. Biochem Biophys Res Commun 171 1312-1318 (1990)
  21. Structural model of the R state of Escherichia coli aspartate transcarbamoylase with substrates bound. Wang J, Eldo J, Kantrowitz ER. J Mol Biol 371 1261-1273 (2007)
  22. Structure of the E.coli aspartate transcarbamoylase trapped in the middle of the catalytic cycle. Stieglitz KA, Dusinberre KJ, Cardia JP, Tsuruta H, Kantrowitz ER. J Mol Biol 352 478-486 (2005)
  23. The 80s loop of the catalytic chain of Escherichia coli aspartate transcarbamoylase is critical for catalysis and homotropic cooperativity. Macol C, Dutta M, Stec B, Tsuruta H, Kantrowitz ER. Protein Sci 8 1305-1313 (1999)
  24. A single amino acid substitution in the active site of Escherichia coli aspartate transcarbamoylase prevents the allosteric transition. Stieglitz KA, Pastra-Landis SC, Xia J, Tsuruta H, Kantrowitz ER. J Mol Biol 349 413-423 (2005)
  25. The first high pH structure of Escherichia coli aspartate transcarbamoylase. Stieglitz KA, Xia J, Kantrowitz ER. Proteins 74 318-327 (2009)
  26. Weakening of the interface between adjacent catalytic chains promotes domain closure in Escherichia coli aspartate transcarbamoylase. Baker DP, Fetler L, Keiser RT, Vachette P, Kantrowitz ER. Protein Sci 4 258-267 (1995)
  27. Intersubunit hydrogen bond acts as a global molecular switch in Escherichia coli aspartate transcarbamoylase. Ha Y, Allewell NM. Proteins 33 430-443 (1998)
  28. Crystal structure of Sulfolobus acidocaldarius aspartate carbamoyltransferase in complex with its allosteric activator CTP. De Vos D, Xu Y, Aerts T, Van Petegem F, Van Beeumen JJ. Biochem Biophys Res Commun 372 40-44 (2008)
  29. The allosteric activator ATP induces a substrate-dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure. Baker DP, Fetler L, Vachette P, Kantrowitz ER. Protein Sci 5 2276-2286 (1996)
  30. Effects of the T-->R transition on the electrostatic properties of E. coli aspartate transcarbamylase. Hariharan M, Allewell NM. Proteins 32 200-210 (1998)
  31. Threonine 82 in the regulatory chain is important for nucleotide affinity and for the allosteric stabilization of Escherichia coli aspartate transcarbamoylase. Williams MK, Kantrowitz ER. Biochim Biophys Acta 1429 249-258 (1998)


Related citations provided by authors (20)

  1. Structural Consequences of Effector Binding to the T State of Aspartate Carbamoyltransferase. Crystal Structures of the Unligated and ATP-, and Ctp-Complexed Enzymes at 2.6-Angstroms Resolution. Stevens RC, Gouaux JE, Lipscomb WN Biochemistry 29 7691- (1990)
  2. Crystal Structures of Aspartate Carbamoyltransferase Ligated with Phosphonoacetamide, Malonate, and Ctp or ATP at 2.8-Angstroms Resolution and Neutral Ph. Gouaux JE, Stevens RC, Lipscomb WN Biochemistry 29 7702- (1990)
  3. Structure of a Single Amino Acid Mutant of Aspartate Carbamoyltransferase at 2.5-Angstroms Resolution. Implications for the Cooperative Mechanism. Gouaux JE, Lipscomb WN, Middleton SA, Kantrowitz ER Biochemistry 28 1798- (1989)
  4. Structural Transitions in Crystals of Native Aspartate Carbamoyltransferase. Gouaux JE, Lipscomb WN Proc. Natl. Acad. Sci. U.S.A. 86 845- (1989)
  5. Complex of N-Phosphonacetyl-L-Aspartate with Aspartate Carbamoyltransferase. X-Ray Refinement, Analysis of Conformational Changes and Catalytic and Allosteric Mechanisms. Ke H, Lipscomb WN, Cho Y, Honzatko RB J. Mol. Biol. 204 725- (1988)
  6. Escherichia Coli Aspartate Transcarbamylase. The Relation between Structure and Function. Kantrowitz ER, Lipscomb WN Science 241 669- (1988)
  7. Three-Dimensional Structure of Carbamoyl Phosphate and Succinate Bound to Aspartate Carbamoyltransferase. Gouaux JE, Lipscomb WN Proc. Natl. Acad. Sci. U.S.A. 85 4205- (1988)
  8. Structural Asymmetry in the Ctp-Liganded Form of Aspartate Carbamoyltransferase from Escherichia Coli. Kim KH, Pan Z, Honzatko RB, Ke H, Lipscomb WN J. Mol. Biol. 196 853- (1987)
  9. 2.5 Angstroms Structure of Aspartate Carbamoyltransferase Complexed with the Bisubstrate Analog N-(Phosphonacetyl)-L-Aspartate. Krause KL, Volz KW, Lipscomb WN J. Mol. Biol. 193 527- (1987)
  10. The Catalytic Mechanism of Escherichia Coli Aspartate Carbamoyltransferase. A Molecular Modelling Study. Gouaux JE, Krause KL, Lipscomb WN Biochem. Biophys. Res. Commun. 142 893- (1987)
  11. Structure at 2.9-Angstroms Resolution of Aspartate Carbamoyltransferase Complexed with the Bisubstrate Analogue N-(Phosphonacetyl)-L-Aspartate. Krause KL, Volz KW, Lipscomb WN Proc. Natl. Acad. Sci. U.S.A. 82 1643- (1985)
  12. Structure of Unligated Aspartate Carbamoyltransferase of Escherichia Coli at 2.6-Angstroms Resolution. Ke H, Honzatko RB, Lipscomb WN Proc. Natl. Acad. Sci. U.S.A. 81 4037- (1984)
  13. Crystal and Molecular Structures of Native and Ctp-Liganded Aspartate Carbamoyltransferase from Escherichia Coli. Honzatko RB, Crawford JL, Monaco HL, Ladner JE, Edwards BFP, Evans DR, Warren SG, Wiley DC, Ladner RC, Lipscomb WN J. Mol. Biol. 160 219- (1982)
  14. Interactions of Phosphate Ligands with Escherichia Coli Aspartate Carbamoyltransferase in the Crystalline State. Honzatko RB, Lipscomb WN J. Mol. Biol. 160 265- (1982)
  15. Interactions of Metal-Nucleotide Complexes with Aspartate Carbamoyltransferase in the Crystalline State. Honzatko RB, Lipscomb WN Proc. Natl. Acad. Sci. U.S.A. 79 7171- (1982)
  16. Gross Quaternary Changes in Aspartate Carbamoyltransferase are Induced by the Binding of N-(Phosphonacetyl)-L-Aspartate. A 3.5-Angstroms Resolution Study. Ladner JE, Kitchell JP, Honzatko RB, Ke HM, Volz KW, Kalb(Gilboa) AJ, Ladner RC, Lipscomb WN Proc. Natl. Acad. Sci. U.S.A. 79 3125- (1982)
  17. A 3.0-Angstroms Resolution Study of Nucleotide Complexes with Aspartate Carbamoyltransferase. Honzatko RB, Monaco HL, Lipscomb WN Proc. Natl. Acad. Sci. U.S.A. 76 5105- (1979)
  18. Three-Dimensional Structures of Aspartate Carbamoyltransferase from Escherichia Coli and of its Complex with Cytidine Triphosphate. Monaco HL, Crawford JL, Lipscomb WN Proc. Natl. Acad. Sci. U.S.A. 75 5276- (1978)
  19. Binding Site at 5.5 Angstroms Resolution of Cytidine Triphosphate, the Allosteric Inhibitor of Aspartate Transcarbamylase from Escherichia Coli. Relation to Mechanisms of Control. Lipscomb WN, Edwards BFP, Evans DR, Pastra-Landis SC STRUCTURE AND CONFORMATION OF NUCLEIC ACIDS AND PROTEIN-NUCLEIC ACID INTERACTIONS : PROCEEDINGS OF THE FOURTH ANNUAL HARRY STEENBOCK SYMPOSIUM, JUNE 16-19, 1974, MADISON, WISCONSIN 333- (1975)
  20. Aspartate Transcarbamoylase from Escherichia Coli. Electron Density at 5.5 Angstroms Resolution. Warren SG, Edwards BFP, Evans DR, Wiley DC, Lipscomb WN Proc. Natl. Acad. Sci. U.S.A. 70 1117- (1973)

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