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 beta-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 K84', which participate in the monomer-monomer interface. Pathway 2 reflects consequent reorganization across this subunit interface, most notably formation of a H74-H74rsquo; pi-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 H74rsquo;. 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.

Figure 1.

(A) Backbone representation of the core domains of LacI dimer in the repressed form (PDB code 1EFA; Bell and Lewis 2000). N-subdomains are in purple; C-subdomains, in green; N-subdomain monomer–monomer interface, in red; flexible loops, in cyan; and interconnecting strands of the core pivot, in pink. Side chains of key residues in the simulation are highlighted in yellow; those that contact inducer are in orange. The chloride anion is represented by a yellow ball. The asterisk locates one of the inducer-binding pockets. The figure was made by using Molscript (Kraulis 1991) and rendered by Povray (www.povray.org). (B) Top views of trigger monomer, illustrating the N-subdomain’ hydrophobic group changes. Time intervals are listed on the top left corner of each structure. In larger representation on the left, residues involved are labeled and indicated with a stick representation. The three smaller figures on the right show the same residues in space-filling representation as they change positions over the course of the simulation. Arrows illustrate direction of movement of each region during the simulation. Counterclockwise rotation about the N-subdomain’ hydrophobic group causes contraction in the center of this region.

Figure 2.

(A) Timeline of the allosteric transition pathway as simulated by TMD. Residues that contact inducer are underlined. Trigger monomer interactions are highlighted in yellow boxes; response monomer interactions, in green; and interactions involving both monomers, in blue. Boxes with dark orange borders are used to delineate pathway 1; pink borders, pathway 2; borders with black dotted lines, pathway 3; pink dotted lines, pathway 2/3; and a black border, involvement in all three pathways. (B) The structures of five representative time points along the simulated trajectory were aligned using the Cα residues of the stationary C-subdomains. The structures are colored as follows: red (0 psec), yellow (60 psec), green (110 psec), blue (160 psec), and purple (210 psec). The flexible loop (residues 149–156) is enclosed by a dotted circle. The directionality of pathway 1 is illustrated by the red arrows; pathway 2, by the blue arrow; and pathway 3, by the black arrow.

Figure 3.

Reproducibility of seven TMD trajectories. The distance changes in the monomer–monomer interface for residues of pathway 2 provide a good example for the repeatability of changes during the simulation. Over the course of the transition, the distance between the side-chains of K84 and K84’ increases (A), whereas the distance between the Cαs of V96 and V96’ (B), and hence β-strands B and B’, decreases. (C) Notice that the squeezing together of the β-strands (V96) coincides with the departure of the lysine pair (K84).

Figure 4.

Interatomic distance versus time plots of hydrogen bonds formed between main chain of D149’ and side chain of S193′ on trigger monomer (A), main chain of D149 and side chain of S193 on response monomer (B), side chain of D149’ and main chain of F161’ on trigger monomer (C), and side chain of D149 and main chain of F161 on response monomer (D). Arrow 1 in A notes time when hydrogen bond forms between side chains of D149’ and S193′ (D149’-OD1 and S193′-HG). Arrow 2 in A illustrates hydrogen bonding between main chain of D149’ and the side chain of S193′ (D149’-O and S193′-HG). Arrow 3 in C marks the beginning of hydrogen bonding between the side chain of D149’ and the main chain of F161’. (E–H) Mobility of the flexible loop (residues 149–156) versus time for both monomers. Distances to the relevant Cα in the flexible loop were measured from a stationary anchor point in the C-subdomains (P284’-Cα and P284-Cα). The plots stabilize near 110 psec, after the N-subdomain interface is closed. (I, J) Hydrogen bond formation between atoms in β-strands C and D versus time. Filled circles indicate the distance between L128-NH and D149-O; open diamonds, Y126-O to D149-NH; filled triangles, I124-O to L148-NH; and plus symbols, I124-NH to L146-O. Notice the break in hydrogen bonding between L128’–D149’ and Y126’–D149’ at 90 psec between β-strands C’ and D’ (arrow 4). The asymmetry at the two endpoints of the graphs is not present in the inducer-bound crystal structure but occurs during the equilibration.

Figure 5.

Schematic representation of N-subdomain monomer–monomer interface in the repressed (A) and the induced (B) conformations. The view is from above the N-subdomains looking down into the interface. Filled circles indicate the side chain is above the plane of the β-sheets; open circles, the side chain lies below the plane. Note that residue 84 is in the plane of the β-sheets, even though its side chain is white. The repressed interface (A) is dominated by hydrogen bond and electrostatic interactions (dotted lines) that stabilize the K84 pair and chloride anion in the space located between the two β-strands. In the induced conformation (B), the K84 side chains are outside the interface, allowing the backbones of V96 and V96’ to form hydrogen bonds, creating an antiparallel β-sheet across the interface. V94 and V96 are also members of the N-subdomain hydrophobic group (see Fig. 1B ▶).

Figure 6.

Interatomic distance (A, C, E) and dihedral angle (B, D, F) versus time plots for H74 and H74’ for representative trajectories (1, 5, and 7). (B, D, F) The H74 dihedral angle of the trigger monomer is represented by circles; that for the response monomer, by the solid line. In order for stable parallel π-stacking to occur, the histidine side chains must have a similar dihedral angle (indicating that the rings are oriented in parallel planes), and the rings must be ∼4.5 Å apart (shown as dashed line in A,C, E). These conditions first exist concurrently in trajectory 1 at 110 psec (arrow 1), in trajectory 5 at 95 psec (arrow 4), and in trajectory 7 at 95 psec (arrow 7). The two histidines undergo simultaneous 180° rotations in trajectory 1 at 170 psec (arrow 2), in trajectory 5 at 140 psec (arrow 5), and in trajectory 7 at 140 psec (arrow 8), which allows them to reform π-stacking until they adopt their final conformations. The noise following arrows 3 and 6 is due to His74’ adopting the equilibrated inducer-bound target structure in the simulation, in which π-stacking does not exist.

Figure 7.

(A) Detailed view of the core pivot hydrophobic group in the trigger monomer. Interactions between residues are centered on F161’. (B) Backbone φ and ξ plots versus time for core pivot residues in trigger monomer. All residues plotted have standard deviations >9°, with the exception of P320’-Phi, which is displayed for comparison. Dotted lines indicate the approximate average angles at the start of the simulation. Arrows call attention to significant changes in φ/ξ angle.

Figure 8.

Interatomic distance versus time plots between F293 and F161, two residues in the core pivot hydrophobic cluster. The dotted line represents the approximate average distance at the start of the simulation and aids visual inspection. F293’ moves toward F161’ at 110 psec, whereas F293 moves closer to F161 at the same time. The asymmetry of the two endpoints is the result of asymmetry from the original crystal structure.