Atomic level description of the domain closure in a dimeric enzyme

Thermus thermophilus 3-isopropylmalate dehydrogenase

Éva Gráczer, Angelo Merli, Rajesh Kumar Singh, Manikandan Karuppasamy, P. Závodszky, Manfred S. Weiss, M. Vas

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The domain closure associated with the catalytic cycle is described at an atomic level, based on pairwise comparison of the X-ray structures of homodimeric Thermus thermophilus isopropylmalate dehydrogenase (IPMDH), and on their detailed molecular graphical analysis. The structures of the apo-form without substrate and in complex with the divalent metal-ion to 1.8 Å resolution, in complexes with both Mn2+ and 3-isopropylmalate (IPM), as well as with both Mn2+ and NADH, were determined at resolutions ranging from 2.0 to 2.5 Å. Single crystal microspectrophotometric measurements demonstrated the presence of a functionally competent protein conformation in the crystal grown in the presence of Mn2+ and IPM. Structural comparison of the various complexes clearly revealed the relative movement of the two domains within each subunit and allowed the identification of two hinges at the interdomain region: hinge 1 between αd and βF as well as hinge 2 between αh and βE. A detailed analysis of the atomic contacts of the conserved amino acid side-chains suggests a possible operational mechanism of these molecular hinges upon the action of the substrates. The interactions of the protein with Mn2+ and IPM are mainly responsible for the domain closure: upon binding into the cleft of the interdomain region, the substrate IPM induces a relative movement of the secondary structural elements βE, βF, βG, αd and αh. A further special feature of the conformational change is the movement of the loop bearing the amino acid Tyr139 that precedes the interacting arm of the subunit. The tyrosyl ring rotates and moves by at least 5 Å upon IPM-binding. Thereby, new hydrophobic interactions are formed above the buried isopropyl-group of IPM. Domain closure is then completed only through subunit interactions: a loop of one subunit that is inserted into the interdomain cavity of the other subunit extends the area with the hydrophobic interactions, providing an example of the cooperativity between interdomain and intersubunit interactions.

Original languageEnglish
Pages (from-to)1646-1659
Number of pages14
JournalMolecular BioSystems
Volume7
Issue number5
DOIs
Publication statusPublished - May 1 2011

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3-Isopropylmalate Dehydrogenase
Thermus thermophilus
Enzymes
Hydrophobic and Hydrophilic Interactions
Amino Acids
Protein Conformation
NAD
beta-isopropylmalate
Oxidoreductases
Metals
X-Rays
Ions

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology

Cite this

Atomic level description of the domain closure in a dimeric enzyme : Thermus thermophilus 3-isopropylmalate dehydrogenase. / Gráczer, Éva; Merli, Angelo; Singh, Rajesh Kumar; Karuppasamy, Manikandan; Závodszky, P.; Weiss, Manfred S.; Vas, M.

In: Molecular BioSystems, Vol. 7, No. 5, 01.05.2011, p. 1646-1659.

Research output: Contribution to journalArticle

Gráczer, Éva ; Merli, Angelo ; Singh, Rajesh Kumar ; Karuppasamy, Manikandan ; Závodszky, P. ; Weiss, Manfred S. ; Vas, M. / Atomic level description of the domain closure in a dimeric enzyme : Thermus thermophilus 3-isopropylmalate dehydrogenase. In: Molecular BioSystems. 2011 ; Vol. 7, No. 5. pp. 1646-1659.
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abstract = "The domain closure associated with the catalytic cycle is described at an atomic level, based on pairwise comparison of the X-ray structures of homodimeric Thermus thermophilus isopropylmalate dehydrogenase (IPMDH), and on their detailed molecular graphical analysis. The structures of the apo-form without substrate and in complex with the divalent metal-ion to 1.8 {\AA} resolution, in complexes with both Mn2+ and 3-isopropylmalate (IPM), as well as with both Mn2+ and NADH, were determined at resolutions ranging from 2.0 to 2.5 {\AA}. Single crystal microspectrophotometric measurements demonstrated the presence of a functionally competent protein conformation in the crystal grown in the presence of Mn2+ and IPM. Structural comparison of the various complexes clearly revealed the relative movement of the two domains within each subunit and allowed the identification of two hinges at the interdomain region: hinge 1 between αd and βF as well as hinge 2 between αh and βE. A detailed analysis of the atomic contacts of the conserved amino acid side-chains suggests a possible operational mechanism of these molecular hinges upon the action of the substrates. The interactions of the protein with Mn2+ and IPM are mainly responsible for the domain closure: upon binding into the cleft of the interdomain region, the substrate IPM induces a relative movement of the secondary structural elements βE, βF, βG, αd and αh. A further special feature of the conformational change is the movement of the loop bearing the amino acid Tyr139 that precedes the interacting arm of the subunit. The tyrosyl ring rotates and moves by at least 5 {\AA} upon IPM-binding. Thereby, new hydrophobic interactions are formed above the buried isopropyl-group of IPM. Domain closure is then completed only through subunit interactions: a loop of one subunit that is inserted into the interdomain cavity of the other subunit extends the area with the hydrophobic interactions, providing an example of the cooperativity between interdomain and intersubunit interactions.",
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