Probing the transient dark state of substrate binding to GroEL by relaxation-based solution NMR

Abstract

The mechanism whereby the prototypical chaperonin GroEL performs work on substrate proteins has not yet been fully elucidated, hindered by lack of detailed structural and dynamic information on the bound substrate. Previous investigations have produced conflicting reports on the state of GroEL-bound polypeptides, largely due to the transient and dynamic nature of these complexes. Here, we present a unique approach, based on combined analysis of four complementary relaxation-based NMR experiments, to probe directly the "dark" NMR-invisible state of the model, intrinsically disordered, polypeptide amyloid β (Aβ40) bound to GroEL. The four NMR experiments, lifetime line-broadening, dark-state exchange saturation transfer, relaxation dispersion, and small exchange-induced chemical shifts, are dependent in different ways on the overall exchange rates and populations of the free and bound states of the substrate, as well as on residue-specific dynamics and structure within the bound state as reported by transverse magnetization relaxation rates and backbone chemical shifts, respectively. Global fitting of all the NMR data shows that the complex is transient with a lifetime of <1 ms, that binding involves two predominantly hydrophobic segments corresponding to predicted GroEL consensus binding sequences, and that the structure of the bound polypeptide remains intrinsically and dynamically disordered with minimal changes in secondary structure propensity relative to the free state. Our results establish a unique method to observe NMR-invisible dynamic states of GroEL-bound substrates and to describe at atomic resolution the events between substrate binding and encapsulation that are crucial for understanding the normal and stress-related metabolic function of chaperonins.

Keywords: conformational sampling; protein–protein interactions; supramolecular machine.

Fig. 1.

¹⁵N-ΔR₂, ¹⁵N-DEST, and ¹⁵N relaxation dispersion profiles of Aβ40 in the presence of GroEL. (A) ¹⁵N-ΔR₂ (CPMG field = 550 Hz) for 50 μM ¹⁵N-labeled Aβ40 in the presence of 20 (circles) and 40 (diamonds) μM (in subunits) GroEL at spectrometer frequencies of 600 (blue) and 900 (red) MHz. ¹⁵N-ΔR₂ at 600 MHz for a control sample (green squares) containing 50 μM ¹⁵N-labeled Aβ40, 2.9 μM acid-denatured Rubisco, and 20 μM GroEL [in subunits, corresponding to 2.9 μM in Rubisco binding sites (i.e., one binding site per GroEL cavity)] demonstrates that acid-denatured Rubisco displaces GroEL-bound Aβ40. (B) Comparison of observed (obs) ¹⁵N-ΔR₂ values with the calculated (calc) values obtained from global fitting of all experimental data to a two-state exchange model. (C) Examples of ¹⁵N-DEST profiles (plotted as normalized cross-peak intensities as a function of frequency offset from the ¹⁵N carrier at 118.5 ppm) obtained with rf (RF) field strengths of 250 (open circles) and 500 (closed circles) Hz for the ¹⁵N continuous wave saturation pulse recorded at a spectrometer frequency of 900 MHz on a sample containing 50 μM ¹⁵N-labeled Aβ40 in the presence of 20 μM (in subunits) GroEL. (D) ¹⁵N-CPMG relaxation dispersion curves at spectrometer frequencies of 600 (blue) and 900 (red) MHz observed for 50 μM ¹⁵N-labeled Aβ40 in the presence of 20 μM (in subunits) GroEL. Reference dispersion curves at 600 MHz for ¹⁵N-labeled Aβ40 in the absence of GroEL are shown in green. The red and blue dashed and solid lines in A and D are the best-fit curves obtained by simultaneously fitting all the experimental data to a two-state exchange model. The green lines in A and D serve to guide the eye. The sequence of Aβ40 is shown at the top of the figure with hydrophobic residues highlighted in green and the GroEL substrate consensus sequences (where P stands for polar residues and H stands for hydrophobic residues) (28) aligned above the Aβ40 sequence for reference. All experiments were conducted at 5 °C. Error bars = 1 SD.

Fig. 2.

Observed exchange-induced chemical shifts for Aβ40 in the presence of GroEL. (A) ¹⁵N, ¹H_N, ¹³Cα, and ¹³Cβ chemical shift differences (in hertz) between 50 μM ¹⁵N-labeled Aβ40 samples containing 20 and 0 μM GroEL at a spectrometer frequency of 900 MHz. (B) Expansions of selected regions of the ¹H-¹⁵N HSQC spectra and ¹H-¹³C constant time HSQC spectra of 50 μM ¹⁵N-labeled Aβ40 in the absence (black contours) and presence (red contours) of 20 μM GroEL. Correlation of ¹⁵N exchange-induced chemical shifts of ¹⁵N-labeled Aβ40 at two spectrometer frequencies (C) and two GroEL concentrations (D) is shown. corr. coeff., correlation coefficient. (E) ¹⁵N-ΔR₂ values are not correlated to the ¹⁵N exchange-induced chemical shifts. (F) Comparison of observed ¹⁵N exchange-induced shifts with the calculated values obtained by simultaneously fitting all the experimental data to a two-state exchange model. Error bars = 1 SD.

Fig. 3.

Kinetic scheme for Aβ40 binding to GroEL. (A) Two-state exchange model describing the association of Aβ40 with GroEL. The values listed for and the populations of free (p_A) and GroEL-bound (p_B) Aβ40 are those obtained in the presence of 20 μM GroEL. The equilibrium dissociation constant K_d and the second-order association rate constant k_on are calculated assuming each GroEL cavity only accommodates a single molecule of Aβ40 with numerous available binding modes. (B) Rapid interconversion (with a rate constant >k_off) between different GroEL-bound configurations of Aβ40 consisting of the central hydrophobic, C-terminal hydrophobic, or both hydrophobic regions in contact with GroEL is possible.

Fig. 4.

Calculated residue-specific parameters for Aβ40 in the dark GroEL-bound state. (A) Optimized values of the residue-specific backbone ¹⁵N-R₂ rates, , for GroEL-bound Aβ40 at spectrometer frequencies of 600 (blue) and 900 (red) MHz. (B) Residue-specific ¹⁵N, ¹H_N, ¹³Cα, and ¹³Cβ chemical shift differences between GroEL-bound and free Aβ40. The ¹⁵N chemical shift differences (¹⁵N-Δδ) are optimized in the fitting procedure, whereas the other chemical shift differences are calculated from the ΔR₂ values and fitted global kinetic parameters (SI Materials and Methods). (C) Secondary structure populations (top, coil; middle, β and polyproline II; bottom, α) for free (blue line) and GroEL-bound (red circles) Aβ40 (Left, y axis) obtained using the δ2D method (29) and the difference in secondary structure populations between bound and free Aβ40 (gray bars; Right, y axis). Error bars = 1 SD.