The folding energy landscape of the dimerization domain of Escherichia coli Trp repressor: a joint experimental and theoretical investigation

Abstract

Enhanced structural insights into the folding energy landscape of the N-terminal dimerization domain of Escherichia coli tryptophan repressor, [2-66]2 TR, were obtained from a combined experimental and theoretical analysis of its equilibrium folding reaction. Previous studies have shown that the three intertwined helices in [2-66]2 TR are sufficient to drive the formation of a stable dimer for the full-length protein, [2-107]2 TR. The monomeric and dimeric folding intermediates that appear during the folding reactions of [2-66]2 TR have counterparts in the folding mechanism of the full-length protein. The equilibrium unfolding energy surface on which the folding and dimerization reactions occur for [2-66]2 TR was examined with a combination of native-state hydrogen exchange analysis, pepsin digestion and matrix-assisted laser/desorption mass spectrometry performed at several concentrations of protein and denaturant. Peptides corresponding to all three helices in [2-66]2 TR show multi-layered protection patterns consistent with the relative stabilities of the dimeric and monomeric folding intermediates. The observation of protection exceeding that offered by the dimeric intermediate in segments from all three helices implies that a segment-swapping mechanism may be operative in the monomeric intermediate. Protection greater than that expected from the global stability for a single amide hydrogen in a peptide from the C-helix possibly and another from the A-helix may reflect non-random structure, possibly a precursor for segment swapping, in the urea-denatured state. Native topology-based model simulations that correspond to a funnel energy landscape capture both the monomeric and dimeric intermediates suggested by the HX MS data and provide a rationale for the progressive acquisition of secondary structure in their conformational ensembles.

Figure 1

Crystal structure of wild-type E. coli Trp repressor. The two monomers are displayed in blue and gray. The six helices in each monomer are denoted A-F. The core region, [2-66]₂ TR, which is comprised of the first three helices of each monomer, A, B and C, ends at residue 66, the position of which is indicated.

Figure 2

Peptide coverage map of [2-66]₂ TR. The eight peptides whose HX data were processed are shown as double-headed arrows underneath the amino acid sequence for [2-66]₂ TR. The location of the A, B and C helices are shown above the amino acid sequence. Dashed arrows indicate segments that were analyzed by subtracting the masses of two peptides from each other.

Figure 3

Time-dependent shift of the isotopic envelope for peptide 26-35 in 10 mM phosphate at pH 7.2 and 25 °C in D₂O. The progressive shift of the isotopic envelope to higher mass/charge (m/z) ratio for the +1 ion demonstrates EX2 exchange kinetics.

Figure 4

Graphical interpretation of the data-fitting routine for peptide 42-61 obtained by incubating 20 μM [2-66]₂ TR in 10 mM phosphate, pH 7.2, and 25 °C in the absence of denaturant for up to 4 weeks in D₂O. Amide hydrogens exchanging via the fast reaction, k_f, are quantified by extrapolating the exponential fits back to 1 s. The other three exponentials, k_i1, k_i2, and ks, are indicated.

Figure 5

(A) Urea dependence of the HX properties of peptide 42-61. Urea concentrations for exchange were 0 M (circles), 0.50 M (triangles) and 1.00 M (squares). (B) Protein concentration dependence of the HX properties of peptide 42-61. Protein concentrations for exchange were 10 μM (squares), 20 μM (triangles) and 50 μM (circles). The log time coordinate enables the display of the full data set out to 24 hours. The inflections highlight the various exchange regimes. Exchange conditions were 10 mM phosphate, pH 7.2, and 25 °C in D₂O.

Figure 6

The folding/association mechanism of the [8-66]₂ Trp repressor from Gō and symmetrized-Gō models. (A) A typical trajectory of the folding of [8-66]₂ TR simulated with the symmetrized-Gō model. The trajectory illustrates the unfolded and folded states (U and N₂, respectively) and the on pathway dimeric intermediate (I₂). Similar trajectories are observed when simulating using the Gō model. The monomeric intermediate (I) is not seen in this trajectory as it is relatively unstable around the folding temperature. (B) and (C) show 4-dimensional free energy surfaces for folding and association of [8-66]₂ TR at the folding temperature using Gō and symmetrized-Gō models, respectively. The reaction coordinates in this free energy plot are the number of contacts of a single subunit (i.e., monomer folding), the number of contacts in the dimer interface (i.e., association), and the separation distance between the two subunits.

Figure 7

The dimeric intermediate in the folding/association of [8-66]₂ TR obtained from the native topology-based models. (A) Free energy surface of the folding of [8-66]₂ TR projected on two reaction coordinates. The first reaction coordinate measures the interfacial contacts between helices A, B, A’, and B’ and the second measures all the interfacial contacts with helices C and C’. It indicates that in the dimeric intermediate helix C and C’ are not interacting intermolecularly. (B) The contact probability map for the dimeric intermediate. The upper and lower halves respectively show the contact probabilities of the dimeric intermediate from the Gō and symmetrized-Gō models. The flexibility of helices C and C’ is indicated by the lower probabilities for the interfacial contacts that involved the residues of helices C and C’.

Figure 8

The monomeric intermediate in the folding/association of [8-66]₂ TR obtained from the native topology-based models. (A) Free energy surface of the folding of [8-66]₂ TR projected on the native contact of the monomer (as defined in the Gō model) and the monomeric non-native contacts (as defined in the symmetrized-Gō model). This plot shows that an almost fully folded monomer can be found with a large amount of non-native contacts that result in packing of the α-helices. (B) The contact probability at the folded (F) and unfolded (U) state of monomeric TR (residues 8-66) from simulations of an isolated monomer using the symmetrized-Gō potential. (C) A typical structure of folded monomeric [8-66] TR.

Figure 9

Free energy diagram for the folding of [2-66]₂ TR. Four states are proposed to exist, the native dimer, N₂, a dimeric intermediate, I₂, a monomeric intermediate, I, and the unfolded monomer, U. Solid lines indicate the relative free energies at equilibrium and at 20 μM [2-66]₂ TR as determined previously., Gray bars indicate the average protection factors and implied free energies obtained for each amide hydrogen exchange rate averaged over all peptides under exchange conditions of 20 μM protein.

Figure 10

Histograms of the relative protection factors of the residues comprising helices A, B, and C in the monomeric and dimeric intermediates, I and I₂. The protection factors are estimated from the native topology-based model simulations supplemented by swapped/unswapped interhelical interactions. The protection factor was assumed to be proportional to the number of interactions that any residue forms in any state (Eq. 5).

Figure 11

The relative protection factors in the monomeric, I (left), and dimeric, I₂ (right) intermediates of [8-66]₂ TR as calculated from the native topology-based simulations. Relative protection factors lower than 0.2 or higher than 0.7 are colored orange and gray, respectively. The dimeric intermediate displays much more protection than the monomeric intermediate (the flexible termini in I have high relative protection factors but their absolute protection factors are low in comparison to the helices). The relative protection factors illustrate the interactions between helices A and C and the strain in helix B in state I as well as the stabilization of the dimeric interface between helices A, A’, B, and B’ in state I₂.