Allosteric transition pathways in the lactose repressor protein core domains: Asymmetric motions in a homodimer

Abstract

The crystal structures of lactose repressor protein (LacI) provide static endpoint views of the allosteric transition between DNA-and IPTG-bound states. To obtain an atom-by-atom description of the pathway between these two conformations, motions were simulated with targeted molecular dynamics (TMD). Strikingly, this homodimer exhibited asymmetric dynamics. All asymmetries observed in this simulation are reproducible and can begin on either of the two monomers. Asymmetry in the simulation originates around D149 and was traced back to the pre-TMD equilibrations of both conformations. In particular, hydrogen bonds between D149 and S193 adopt a variety of configurations during repetitions of this process. Changes in this region propagate through the structure via noncovalent interactions of three interconnected pathways. The changes of pathway 1 occur first on one monomer. Alterations move from the inducer-binding pocket, through the N-subdomain ␤-sheet, to a hydrophobic cluster at the top of this region and then to the same cluster on the second monomer. These motions result in changes at (1) side chains that form an interface with the DNA-binding domains and (2) K84 and K84Ј, which participate in the monomer-monomer interface. Pathway 2 reflects consequent reorganization across this subunit interface, most notably formation of a H74-H74Ј -stacking intermediate. Pathway 3 extends from the rear of the inducer-binding pocket, across a hydrogen-bond network at the bottom of the pocket, and transverses the monomer-monomer interface via changes in H74 and H74Ј. In general, intermediates detected in this study are not apparent in the crystal structures. Observations from the simulations are in good agreement with biochemical data and provide a spatial and sequential framework for interpreting existing genetic data.Keywords: Gene regulation; allosteric mechanism; structural flexibility; conformational transition pathway; targeted molecular dynamics Supplemental material: See www.proteinscience.org Since its discovery in 1961, the lactose (lac) operon has been the prototypic model for studying genetic and allosteric regulation (Jacob and Monod 1961;Monod et al. 1965;Matthews and Nichols 1998;Matthews et al. 2000). The central component of this regulatory system-lactose repressor protein (LacI)-binds to three operator sites within the lac operon to repress gene transcription (Matthews 1992). Inducer sugars, such as allolactose (Jobe and Bourgeois 1972) These authors made equal contributions to this work. Article and publication are at http://www.proteinscience.org/cgi

Structural Analysis of Lac Repressor Bound to Allosteric Effectors

“…12 This mechanism is also consistent with the targeted molecular dynamic studies conducted by Matthews et al that mapped out the steps the repressor takes during the conformational transition from the induced to repressed state. 21 One of the first steps in the transition is the formation of a hydrogen bond between residue D149 of the N-terminal sub-domain and S-193 of the C-terminal sub-domain. 21 These two residues are both directly involved in the hydrogen bonding network mediated by the O6 hydroxyl found in IPTG, suggesting that the O6 hydroxyl is critical in aiding one of the first steps in transmitting the allosteric signal through the N-terminal sub-domain to the DNA binding domain.…”

“…21 One of the first steps in the transition is the formation of a hydrogen bond between residue D149 of the N-terminal sub-domain and S-193 of the C-terminal sub-domain. 21 These two residues are both directly involved in the hydrogen bonding network mediated by the O6 hydroxyl found in IPTG, suggesting that the O6 hydroxyl is critical in aiding one of the first steps in transmitting the allosteric signal through the N-terminal sub-domain to the DNA binding domain. Miller also demonstrated that D149 and S193 are critical residues, since almost all mutations at either position resulted in a repressor that could no longer respond to inducer.…”

Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins

“…Changes at this pivot occur when small, allosteric ligands bind the regulatory domain. Binding therefore alters the juxtaposition of the N-subdomains, which "pulls" the hinge helices and provides a key mechanism for altering their orientation and contacts to DNA (14,(21)(22)(23).…”

“…For E. coli LacI, structures of free, DNA-bound, and IPTG-bound protein (14,70), along with molecular dynamics simulations (23,71,72), have been used to study these adaptable regions. The largest changes are found in the linker region and in the N-subdomain interface of LacI (14,23,70). Inducer binding alters the juxtaposition of the LacI N-subdomains to bring them into closer contact (Fig.…”

Altering Residues N125 and D149 Impacts Sugar Effector Binding and Allosteric Parameters in Escherichia coli Lactose Repressor

“…Numerous residues are actively involved in transmitting the allosteric signal between these sites (11-15). Crystallographic structures demonstrated that D149 directly contacts the inducer ligand IPTG, and targeted molecular dynamics (TMD) simulation to follow changes upon inducer binding identified residues in the allosteric pathway, including interactions between D149/S193 and D149/N125 (11-13). Mutation to allow disulfide formation between D149C and S193C demonstrated the importance of flexibility between these residues for allosteric response (14).…”