Multiple Conformations of Gal3 Protein Drive the Galactose-Induced Allosteric Activation of the GAL Genetic Switch of Saccharomyces cerevisiae. Issue 1 (6th January 2017)

• Record Type: Journal Article
• Authors: Kar, Rajesh Kumar; Kharerin, Hungyo; Padinhateeri, Ranjith; Bhat, Paike Jayadeva
• Is Part Of: Journal of molecular biology. Volume 429:Issue 1(2017)
• Page Start: 158
• Page End: 176
• Publication Date: 2017-01-06
• Subjects: KNF Koshland–Nemethy–Filmer -- MWC Monod–Wyman–Changeux -- Gal3p Gal3 protein -- MD molecular dynamics -- d.o. drop out -- gly/lac glycerol plus lactate -- 2-DG 2-deoxy-galactose -- Gal1p galactokinase -- ORF open reading frame -- epPCR error-prone PCR -- EtBr ethidium bromide -- SDM site-directed mutagenesis -- FACS fluorescence-activated cell sorting -- CMD conventional MD -- TMD targeted MD -- SASA solvent-accessible surface area -- PCA principal component analysis -- PC1 first principal components -- 3-AT 3-amino-1, 2, 4-triazole -- PBS phosphate-buffered saline -- GFP green fluorescent protein -- VMD visual molecular dynamics; population shift -- galactose signaling -- conformational heterogeneity -- allostery -- molecular dynamics simulations; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2016.11.005
• Languages: English
• ISSNs: 0022-2836

Abstract

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. Graphical Abstract: Highlights: Gal3p activates GAL genetic switch of Saccharomyces cerevisiae . Mechanism of allosteric transition of Gal3p is proposed. A rare super-open conformation of Gal3p is observed. Residues that stabilize super-open conformation are identified. Conformational selection in Gal3p drives the activation of GAL genetic switch.