Multiple Conformations of Gal3 Protein Drive the Galactose Induced Allosteric Activation of the GAL Genetic Switch of Saccharomyces cerevisiae

Gal3p is an allosteric monomeric protein which activates the GAL genetic switch of Saccharomyces cerevisiae in response to galactose. Expression of constitutive mutant of Gal3p or over-expression 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 has indicated that Gal3p exists in open (inactive) and closed (active) conformations. However, mutant of Gal3p that predominantly exists in inactive conformation and 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 over-expression fails to activate the GAL switch on its own, but activates the switch in response to galactose. Over-expression 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. Further, 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 our study and what is known in human glucokinase, we suggest that the above mechanism could be a general theme in causing the allosteric transition.