title: Computational and experimental investigation of constitutive behavior in AraC.
abstract: Many mutations in the N-terminal arm of AraC result in constitutive behavior in which transcription of the araBAD genes occurs even in the absence of arabinose. To begin to understand the mechanism underlying this class of mutations, we used molecular dynamics with self-guided Langevin dynamics to simulate (1) wild-type (WT) AraC, (2) known constitutive mutants resulting from alterations in the regulatory arm, particularly alanine and glycine substitutions at residue 8 because P8G is constitutive, whereas P8A behaves like wild type, and (3) selected variant AraC proteins containing alterations in the dimerization core. In all of the constitutive arm mutants, but not the WT protein, residues 37-42, which are located in the core of the dimerization domain, became restructured. This raised the question of whether or not these structural changes are an obligatory component of constitutivity. Using molecular dynamics, we identified alterations in the core that produced a similar restructuring. The corresponding mutants were constructed and their ara constitutivity status was determined experimentally. Because the core mutants were not found to be constitutive, we conclude that restructuring of core residues 37-42 does not, itself, lead to constitutivity of AraC. The available data lead to the hypothesis that the interaction of the N-terminal arm with something other than the front lip is the primary determinant of the inducing versus repressing state of AraC.
authors.affiliation: Physics Department, Loyola University Maryland, Baltimore, Maryland.
authors.forename: Mary
authors.initials: M
authors.lastname: Lowe
authors.forename: David
authors.initials: D
authors.lastname: Gullotti
authors.forename: Ana
authors.initials: A
authors.lastname: Damjanovic
authors.forename: Ann
authors.initials: A
authors.lastname: Cheng
authors.forename: Stephanie
authors.initials: S
authors.lastname: Dirla
authors.forename: Robert
authors.initials: R
authors.lastname: Schleif
mesh_terms: D000409:Alanine; D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D051777:AraC Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001089:Arabinose / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D030562:Databases, Protein; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005998:Glycine; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009876:Operon; D011392:Proline; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs
chemical_list: D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D029968:Escherichia coli Proteins; D011392:Proline; D001089:Arabinose; D000409:Alanine; D005998:Glycine
keywords: AraC; allostery; cluster; gene regulation; self-guided Langevin dynamics