High-Resolution Mapping of the Folding Transition State of a WW Domain

Abstract

Fast-folding WW domains are among the best-characterized systems for comparing experiments and simulations of protein folding. Recent microsecond-resolution experiments and long duration (totaling milliseconds) single-trajectory modeling have shown that even mechanistic changes in folding kinetics due to mutation can now be analyzed. Thus, a comprehensive set of experimental data would be helpful to benchmark the predictions made by simulations. Here, we use T-jump relaxation in conjunction with protein engineering and report mutational Φ-values (Φ(M)) as indicators for folding transition-state structure of 65 side chain, 7 backbone hydrogen bond, and 6 deletion and /or insertion mutants within loop 1 of the 34-residue hPin1 WW domain. Forty-five cross-validated consensus mutants could be identified that provide structural constraints for transition-state structure within all substructures of the WW domain fold (hydrophobic core, loop 1, loop 2, β-sheet). We probe the robustness of the two hydrophobic clusters in the folding transition state, discuss how local backbone disorder in the native-state can lead to non-classical Φ(M)-values (Φ(M) > 1) in the rate-determining loop 1 substructure, and conclusively identify mutations and positions along the sequence that perturb the folding mechanism from loop 1-limited toward loop 2-limited folding.

Keywords: WW domain; folding transition state; laser T-jump; protein folding; Φ-value analysis.

Figure 1. hPin1 WW structure and native state interactions

(A) Structural cartoon of the hPin1 WW fold, highlighting the two hydrophobic clusters that protrude from either side of the three-stranded β sheet. The individual β strand are color coded blue, while the loop segments and the N- and C-terminal extensions are shown in grey. Side chain contacts that constitute the hydrophobic clusters are shown as van der Waals surfaces. (B) C_α-backbone representation of the three-stranded β sheet region (residues W11-W34), highlighting the ten backbone hydrogen bonds that connect the three β strands and stabilize the 3-stranded β sheet topology. Hydrogen bonds that were perturbed by amine-to-ester mutations for Φ_M analysis are labeled in red. Residues are labeled in single letter code and are numbered. (C) Quantitative analysis of a complete Ala-scan, replacing each of the 33 non-Alanine residues individually with Ala. Destabilizations calculated at 55 °C range from near zero to ~ 9 kJ/mole and are mapped onto the C_α-backbone structure of the folded protein. Four Ala-mutants (labeled black) were either completely or significantly unfolded, even at low temperature (4 °C). For these four mutants, ΔΔG must exceed 9 kJ/mol, but no accurate thermodynamic data can be derived in aqueous buffer without invoking stabilizing co-solvents.

Figure 2. Φ_M-value analysis at 55 °C

(A) Plot of the Φ_M value vs the difference in free energy between wild type and mutant (ΔΔG, in kJ/mol) for β strand (filled red circles) and hydrophobic cluster 1 mutants (filled black circles). (B) Plot of the Φ_M value vs the difference in free energy between wild type and mutant (ΔΔG, in kJ/mol) of loop 1 (filled blue circles) and loop 2 mutants (filled green circles). Errors in Φ_M that exceed the symbol size are shown explicitly. For clarity, individual Φ_M-values are labeled with single letter code. Raw data used to render the plots are provided in Table 1.

Figure 3. Φ_T analysis at 55 °C

Plot of the Φ_T value for wild type hPin1 WW and mutants thereof vs the change in free energy (ΔΔG, in kJ/mol) between wild type and mutant. Φ_T values are calculated using the T₀-fitting procedure (for details, see SI supporting text 1). Φ_T values of side chain and backbone hydrogen bond mutants are color coded red and blue, respectively. Except the obvious five outliers (mutants W11F, T29G, I28N/T29G, N30A, S32s), the Φ_T values are within a ± 25 % error margin of the average Φ_T (0.50, dashed grey horizontal line). The outlier Φ_T values (> 0.70, dotted grey line) are indicative of perturbing mutations that shift the transition state ensemble along the reaction coordinate closer to the native state. Mutational Φ_M values calculated from these mutants are no longer reliable indicators of the unperturbed “wild type” transition state ensemble, and must be excluded from the consensus Φ_M analysis of hPin1 WW transition state structure.

Figure 4. Analysis of the folding transition state of the hPin1 WW domain

(A) Φ_M values of the 34 single and double mutants (dark grey) and the 5 amide-to-ester backbone hydrogen bonds mutants (light grey) that qualify for Φ_M analysis, and that were used for consensus Φ_M mapping of the folding transition state. (B) Φ_M map of the folding transition state, with Φ_M values for 25 of the 34 residues (single letter representation) mapped onto the C-α backbone structure of the N-terminally truncated folded protein (residues 6–39). Left panel: residues W11-W34 that define the 3-stranded β sheet. Right panel: Residues L7-P37 that includes hydrophobic cluster 1 and the N- and C-terminal extensions. For clarity, Φ_M values were grouped and color-coded (0 < Φ_M < 0.30, blue; 0.3 < Φ_M < 0.6, purple, 0.6 < Φ_M < 0.90, pink; Φ_M > 0.90, red). Residues for which classical hydrophobic deletion mutagenesis yields very high, or negative, Φ_M values that are not supported by other mutations or structural context are color coded black. Residues for which no mutant suitable for Φ_M analysis is available are color coded white. Backbone hydrogen bonds that were studied by amide-to-ester mutagenesis are indicated by arrows (same color code as for side chains). Data used to render the figure are provided in Tables 1 and 2.

Figure 5. Φ_M vs sequence map and Φ_Mvs backbone disorder correlation

(A) Plot of Φ_M values vs. the hPin1 WW sequence used for transition state analysis. Individual side chain Φ_M values are color coded red, while those calculated from backbone hydrogen bond mutants are color-coded blue. The solid red line represents the error-weighted average trend of the side chain Φ_M (see Table 2 for data). (B) Tube plot showing the distribution of thermal B factors from the X-ray crystal structure [47] along the backbone of hPin1 WW domain. (C) Plot of thermal B factors vs. the hPin1 WW sequence, showing a pronounced maximum in loop 1, and a smaller maximum in loop 2. (D) Correlation between Φ_M values and thermal B factors for residues M15-R21 with increased local backbone disorder at 55 °C. Side chain (sc) loop 1 mutants are color coded red and backbone hydrogen bond mutants (hb) are color coded blue. The solid lines represent best fits of the experimental data.

Figure 6. Variation of transition state structure with temperature

(A) Plot of Φ_M (60 °C) vs Φ_M (50 °C). On average, Φ_M values increase by 0.07 units when raising the temperature from 50 °C to 60 °C, suggesting that the transition state overall gains native structure upon heating. (B) Plot of Φ_T (60 °C) vs Φ_T (50 °C). On average, Φ_T values increase by 0.15 units when raising the temperature from 50 °C to 60 °C, suggesting that the transition state becomes more native-like at elevated temperature, consistent with Hammond’s postulate. (C) Plot of the Φ_M (60 °C)/ Φ_M (50 °C)-ratio vs the residue number of the hPin1 WW sequence. Data from individual side chain mutants are color coded red. Data from individual backbone hydrogen bond mutants are color coded blue. The solid red line represents the error-weighted average side chain trend. For clarity, the side chain data of E12 (large errors, see Table 2) are not shown.

Figure 7. Average number of native contacts in the folding transition state

(A) Slope of the ground state free energy (∂ΔG_ground(T)/∂T) of the 39 consensus mutants used for Φ_M analysis (filled red circles, solid black line) or the entire set of single and double mutants (excluding the 6 loop 1 insertion and deletion variants) (filled grey circles, dashed black line) at the midpoint of unfolding (T = T_m, with ΔG_ground(T_m = 0). (B) Corresponding plot as in (A) showing the slope of the free energy of activation (∂ΔG_activated(T)/∂T) at the midpoint of unfolding (T = T_m). The ratio of the two slopes (activated/ground) of ~ 0.70 for the 39 consensus mutants (0.63 for the entire mutant set) suggests that about 70 % of the native contacts are developed in the folding transition state, a value that agrees well with the average calculated from the Φ_M data (Table 2), but that is higher than the average Φ_T value (0.50). The loop 1 insertion and deletion variants that change local changes in backbone topology (filled yellow circles) were excluded from the fit, but their values agree well with the extrapolated fits of the mutants with the 6-residue wild type hPin1 WW loop 1.

Figure 8. Φ_M analysis of hPin1 WW variants with loop 1 deletions or insertions mutations

(A) Loop 1 sequences of the hPin1 WW loop 1 deletions or insertions variants. Wild type residues are numbered and color coded grey. Mutated or deleted residues in the loop deletion variants are color coded red (type-I G-bulge turn) and blue (type-I’ turn), while the inserted Gly residues in the loop 1 insertion mutants are highlighted in orange. (B) Superposition of the high resolution X-ray structures of type-I G-bulge variant FiP (1.90 Å resolution, color coded red, left) and the type-I’ variant 3 (1.50 Å resolution, color coded blue, right) with wild type hPin1 WW structure (1.35 Å resolution, color coded grey). (C) Brønsted plot for folding of the loop 1 variants of hPin1 WW at 60 °C, rendered from the data provided in SI Table 2. Filled red circles: 5-residue type-I G-bulge turn mutants (var1, var2). Filled blue circles: 4-residue type-I’ turn variants (var3, var4). Filled green circles: Cross-validated loop 1 side chain and backbone hydrogen bond mutants (6-residue wild type loop 1 context). Filled orange circles: Gly insertion variants (var5, var6). Filled black circles: Outlier/perturbing mutants. Open light grey circles: Non-loop 1 consensus mutants. The solid black line is the line predicted for Φ_M = 1. (D) Bar plot of Φ_M-values for selected mutants shown in (C). Φ_M values calculated for the redesigned loop 1 variants using wild type hPin1 WW as reference are color coded red (5-residue type-I G-bulge variants) and blue (4-residue type-I’ variants). Φ_M values calculated for variants 2 and 4 in the type-I G-bulge (var1, FiP) and type-I’ context (var3) are shown in light red and light blue, respectively.

Figure 9. Hypothetical “hybrid” Φ_M map for the fast-folding FiP variant of hPin1 WW

Hypothetical side chain Φ_M map (red circles and solid red line) for the fast folding FiP variant of hPin1 WW, rendered with side chain Φ_M values of non-loop 1 mutants measured with wild type hPin1 WW as reference (see Fig. 3, SI Table 2 for details) and the side chain Φ_M value for loop 1 FiP WW variant 2 (loop 1 sequence: SSSGR) measured with FiP as “pseudo wild type” reference (loop 1 sequence: SADGR). As two residues were replaced simultaneously in FiP variant 2 (A18S, D19S, see Fig. 8A), the Φ_M value calculated for variant 2 (Φ_M = 0.94 ± 0.05) was assigned to either mutated residue (labeled by asterisks) in FiP. For residues that are probed by multiple side chain mutations, the error-weighted average Φ_M value is shown (see SI Table 2 for details). Experimentally measured backbone hydrogen bond Φ_M values (filled yellow squares) are those measured for wild type hPin1 WW and are assigned to the two residues that engage in the perturbed hydrogen bond (see SI Table 2 for details). The simulated side chain and backbone hydrogen bond Φ_M values and associated errors are shown in green and blue, respectively and were rendered from Fig. 2E in [14]. Residue numbers correspond to the 33-residue FiP sequence and thus account for the shorter loop 1 substructure (deletion of Arg17 of wild type hPin1 WW).