A Conserved Interdomain Interaction Is a Determinant of Folding Cooperativity in the GST Fold

Abstract

The canonical glutathione transferase (GST) fold found in many monomeric and dimeric proteins consists of two domains that differ in structure and conformational dynamics. However, no evidence exists that the two domains unfold/fold independently at equilibrium, indicating the significance of interdomain interactions in governing cooperativity between domains. Bioinformatics analyses indicate the interdomain interface of the GST fold is large, predominantly hydrophobic with a high packing density explaining cooperative interdomain behavior. Structural alignments reveal a topologically conserved lock-and-key interaction across the domain interface in which a bulky hydrophobic residue ("key") protrudes from the surface of the N-domain and inserts into a pocket ("lock") in the C-domain. To better understand the molecular basis for the contribution of interdomain interactions toward cooperativity within the GST fold in the absence of any influence from quaternary interactions, studies were done with two monomeric GST proteins: Escherichia coli Grx2 (EcGrx2) and human CLIC1 (hCLIC1). Replacing the methionine "key" residue with alanine is structurally nondisruptive, whereas it significantly diminishes the folding cooperativity of both proteins. The loss in cooperativity between domains in the mutants is reflected by a change in the equilibrium folding mechanism from a wild-type two-state process to a three-state process, populating a stable folding intermediate.

Conformational Stability Adaptation of a Double-Stranded RNA-Binding Domain to Transfer RNA Ligand

“…These dsRBDs may also exhibit an intrinsically unstable canonical fold, but in those specific cases, the extensions themselves act as folding effectors or a hydrophobic clamp by strengthening the nucleation site(s) of the canonical fold in trans . In essence, the present work concurs to support the concept that trans and long-range hydrophobic interactions are dominant factors in the cooperativity of protein folding and constitute the major driving force in polypeptide collapse, leading to stable and functional native states. − …”

Role of Individual Histidines in the pH-Dependent Global Stability of Human Chloride Intracellular Channel 1

“…This observation may not be entirely related to histidine-mediated pH sensing, however, and we should be cautious in labeling it as such without first examining the structure of these mutants and the relevant thermodynamics. The formation of a stable intermediate at pH 7.0 in the case of these mutants could also be due to the effect of the mutation on the unfolding pathway of the protein via breakage of specific stabilizing local contacts that are not necessarily influenced by pH as has been observed with a number of other CLIC mutants that have been studied in our laboratory (Legg-E’Silva, unpublished work). However, close examination of the thermodynamic parameters obtained from the unfolding of the histidine mutants provides an interesting scenario that is unlike that of any other CLIC mutant that we have studied.…”

Role of Arginine 29 and Glutamic Acid 81 Interactions in the Conformational Stability of Human Chloride Intracellular Channel 1

“…29 The molecular replacement search model structure for both set of crystals was 3O3T (PDB ID). 30 The models were refined with REFMAC5, 31 and model building was performed with Coot. 32 Solvent molecules were added to the models after several rounds of refinement.…”

“…The structures were solved by molecular replacement using PHASER , implemented in the CCP4i suite of programmes . The molecular replacement search model structure for both set of crystals was 3O3T (PDB ID) . The models were refined with REFMAC5, and model building was performed with Coot .…”