title: Multiple Conformations of Gal3 Protein Drive the Galactose-Induced Allosteric Activation of the GAL Genetic Switch of Saccharomyces cerevisiae.
abstract: Gal3p is an allosteric monomeric protein that activates the GAL genetic switch of Saccharomyces cerevisiae in response to galactose. Expression of constitutive mutant of Gal3p or overexpression of wild-type Gal3p activates the GAL switch in the absence of galactose. These data suggest that Gal3p exists as an ensemble of active and inactive conformations. Structural data have indicated that Gal3p exists in open (inactive) and closed (active) conformations. However, a mutant of Gal3p that predominantly exists in inactive conformation and is yet capable of responding to galactose has not been isolated. To understand the mechanism of allosteric transition, we have isolated a triple mutant of Gal3p with V273I, T404A, and N450D substitutions, which, upon overexpression, fails to activate the GAL switch on its own but activates the switch in response to galactose. Overexpression of Gal3p mutants with single or double mutations in any of the three combinations failed to exhibit the behavior of the triple mutant. Molecular dynamics analysis of the wild-type and the triple mutant along with two previously reported constitutive mutants suggests that the wild-type Gal3p may also exist in super-open conformation. Furthermore, our results suggest that the dynamics of residue F237 situated in the hydrophobic pocket located in the hinge region drives the transition between different conformations. Based on this study, we suggest that conformational selection mechanism is the driving force in the allosteric transition of Gal3p, which may have implications in other signaling pathways involving monomeric proteins.
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India.
authors.forename: Rajesh Kumar
authors.initials: RK
authors.lastname: Kar
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India.
authors.forename: Hungyo
authors.initials: H
authors.lastname: Kharerin
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India.
authors.forename: Ranjith
authors.initials: R
authors.lastname: Padinhateeri
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India. Electronic address: jayadeva@iitb.ac.in.
authors.forename: Paike Jayadeva
authors.initials: PJ
authors.lastname: Bhat
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D005690:Galactose / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: C099289:Gal3 protein, S cerevisiae; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D005690:Galactose
keywords: allostery; conformational heterogeneity; galactose signaling; molecular dynamics simulations; population shift