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Confinement and stabilization of Fyn SH3 folding intermediate mimetics within the cavity of the chaperonin GroEL demonstrated by relaxation-based NMR David S. Libich, Vitali Tugarinov, Rodolfo Ghirlando, and G. Marius Clore Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01237 • Publication Date (Web): 03 Feb 2017 Downloaded from http://pubs.acs.org on February 6, 2017
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Biochemistry
Confinement and stabilization of Fyn SH3 folding intermediate mimetics within the cavity of the chaperonin GroEL demonstrated by relaxation-based NMR David S. Libich,1,‡ Vitali Tugarinov,1,‡ Rodolfo Ghirlando2 and G. Marius Clore1,* Laboratories of Chemical Physics1 and Molecular Biology,2 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520
Supporting Information Placeholder ABSTRACT: The interaction of two folding intermediate
mimetics of the model protein substrate Fyn SH3 with the chaperonin GroEL, a supramolecular foldase/unfoldase machine, has been investigated by 15N relaxation-based NMR spectroscopy (lifetime line broadening, dark state exchange saturation transfer and relaxation dispersion). The two mimetics comprise C-terminal truncations of wild type and triple mutant (A39V/N53P/V55L) Fyn SH3 in which the C-terminal strand of the SH3 domain is unfolded, while preserving the remaining domain structure. Quantitative analysis of the data reveals that a mobile state of the SH3 domain confined and tethered within the cavity of GroEL, possibly through interactions with the disordered, methionine-rich C-terminal tail(s), can be detected, and that the native state of the folding intermediate mimetics is stabilized by both confinement within and binding to apo GroEL. These data provide a basis for understanding the passive activity of GroEL as a foldase/unfoldase: the unfolded state, in the absence of hydrophobic GroELbinding consensus sequences, is destabilized within the cavity due to its larger radius of gyration compared to that of the folding intermediate, while the folding intermediate is stabilized relative to the native state owing to exposure of a hydrophobic patch which favors GroEL binding.
GroEL is a large (800 kDa) supramolecular machine that facilitates protein folding and protects against the damaging effects of misfolding and aggregation.1 GroEL comprises two heptameric rings, each of which encloses a large cavity accessible to protein substrates. The mechanism whereby GroEL exerts its effect encompasses a series of concerted allosteric transitions driven by the hydrolysis of ATP and the binding and dissociation of the co-chaperonin GroES that caps the open rings of GroEL. Apo GroEL, however, can also function as a passive anti-aggregation chamber as
evidenced by both H/D and fluorescence-based folding experiments.2 Recently, we presented a quantitative study of the interaction of a model, metastable SH3 domain with apo GroEL3 by combined analysis of 15N Carr-Purcell-MeiboomGill (CPMG) relaxation dispersion,4 dark state exchange saturation transfer (DEST)5 and lifetime line broadening (ΔR2)6 NMR experiments.7 The model domain consisted of a triple (A39V/N53P/V55L) mutant of Fyn SH3 (SH3Mut) that, at low temperature (10 °C) exists in equilibrium between the major native state (F) and a sparsely-populated (~2%) folding intermediate (I) in which the C-terminal βstrand is disordered, exposing a hydrophobic patch.8 We showed that apo GroEL stabilizes the folding intermediate, accelerates the overall interconversion between the two states ~20-fold, and increases the rate constant for the F to I transition by ~500-fold.3 In this paper, we investigate two mimetics of the folding intermediate, one stable, the other metastable, and present NMR evidence for confinement and stabilization of the folding intermediate mimetics within the cavity of apo GroEL. The two mimetics of the Fyn SH3 folding intermediate comprise three- (SH3WT∆57) and four- (SH3Mut∆56) residue C-terminal truncations of the full-length wild type and triple mutant domains, respectively (see SI). The 1H-15N correlation spectra of SH3Mut∆56 and SH3WT∆57 are well resolved and characteristic of folded proteins (Fig. S1). The global folds for both truncated constructs, determined using CS-Rosetta9 from backbone chemical shifts and residual dipolar couplings (Fig. S2), are very similar to one another (Fig. 1A) as well as to the native and folding intermediate states of the full-length SH3Mut (Fig. S3). The distinguishing characteristic of both truncation mutants and the folding intermediate8 of full length SH3Mut is the unfolding of the Cterminal β-strand; the remaining domain structure, however, remains essentially unchanged relative to the native state. Analytical ultracentrifugation (Fig. S4) and 15N CPMG relaxation dispersion measurements (Fig. S5) show that the
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folded (F) monomeric state of SH3Mut∆56 undergoes concentration-dependent exchange with a folded dimer (FD) and concentration-independent exchange with the fully unfolded (U) state, while SH3WT∆57 is a stable folded monomer with no significant relaxation dispersion (Rex < 1 s-1). For SH3Mut∆56, where large dispersions are observed, the calculated 15N chemical shifts for the unfolded state are fully consistent with those predicted for a random coil (Fig. S5B); in addition, five residues have optimized values > 0.7 ppm for the 15N chemical shift differences between folded monomeric and dimeric states (Fig. S5C). The latter, together with residues showing severely broadened 1H/15N correlations at high concentration of SH3Mut∆56, form a single contiguous dimerization interface that largely coincides with the hydrophobic GroEL-binding surface3 (Fig. S6). C
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Figure 1. Interaction of SH3Mut∆56 with apo GroEL. (A) Ribbon diagrams showing a superposition of the structures of SH3Mut∆56 (blue) and SH3WT∆57 (green), representing mimetics of a folding intermediate of the Fyn SH3 domain, determined from backbone chemical shifts and residual dipolar couplings using CS-Rosetta9 (see SI and Figs. S2 and S3). (B) 15 N ∆R2 observed for 100 µM 15N-labeled SH3Mut∆56 in the presence of 120 µM (in subunits) apo GroEL at 900 (blue circles) and 600 (red circles) MHz and 10°C. The dashed lines represent the best-fits obtained by simultaneously fitting all relaxation data (∆R2, DEST and relaxation dispersion) to the model shown in Fig. 3A. The ∆R2 effect is essentially abolished when the GroEL cavity is blocked by acid-denatured Rubisco (green circles). The grey bar indicates the residues deleted in SH3Mut∆56. The sequence (residues in red are sites of mutations) and secondary structure are shown above the panel; the 310 helix and β5 (dashed outline) are unfolded in SH3Mut∆56, but only β5 is unfolded in SH3WT∆57 (see panel A). Error bars = 1 S.D. (C) Cross-section through apo GroEL (PDB 3E7610) illustrating the two cavities with a SH3Mut∆56 molecule (orange, space-filling model) confined in each cavity.
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(Fig. 1B) and SH3WT∆57 (Fig. S7A) which is largely abolished when the cavity of GroEL is blocked by aciddenatured Rubisco (Fig. 1B), a protein substrate that binds with nanomolar affinity to GroEL.11 Significant broadening of 15N-DEST profiles is also observed for both SH3Mut∆56 (Fig. 2A) and SH3WT∆57 (Fig. S7B) in the presence of apo GroEL with widths at half height of ~5 kHz at saturation field strengths of 500 to 750 Hz. No significant relaxation dispersions are observed for SH3WT∆57 in the presence of apo GroEL indicating that any unfolded GroEL-bound species remains below the level of detection. Thus, one can conclude that the ∆R2 and DEST effects arise from the binding of the folded truncated species to the high molecular weight apo GroEL (presumably at the apical domain). The magnetic field dependence of ΔR2 is smaller than that expected for a relaxation mechanism based on the one-bond 1 H-15N dipolar coupling interaction and a -170 ppm 15N chemical shift anisotropy: !R2900 / !R2600 =1.08±0.08 for SH3Mut∆56 (Fig. 1B) and !R2800 / !R2600 = 1.14±0.06 SH3WT∆57 (Fig. S7A) compared to expected values of 1.3 and 1.2, respectively, indicating that exchange is in the slowto-intermediate exchange regime on the relaxation time scale and the overall value of the pseudo-first order rate constant for exchange between small (observable) and high molecular weight ‘dark’ species is close to the maximum value of ΔR2 .5-6 Normalized intensity
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Figure 2. 15N-DEST and CPMG relaxation dispersion observed for 100 µM 15N-labeled SH3Mut∆56 in the presence of 120 µM (in subunits) apo GroEL at 10°C. (A) Examples of 15N-DEST profiles at RF field strengths of 500 (orange circles) and 750 (purple circles) Hz. (B) Examples of 15N CPMG relaxation dispersion curves in the presence (red circles) and absence (blue circles) of apo GroEL at 900 (top) and 600 (bottom) MHz. The solid lines represent best-fits obtained by simultaneously fitting all relaxation data (∆R2, DEST and relaxation dispersion) to the model shown in Fig. 3.
The 15N-CPMG dispersion profiles for SH3Mut∆56 in the presence of apo GroEL are characterized by a reduction in Rex (i.e. the difference in R2,eff between the lowest and highest CPMG field strengths) relative to those for free SH3Mut∆56
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Biochemistry
(Fig. 2B). This is manifested by only a small increase in R2,eff at the lowest CPMG field while at high CPMG fields (1000 Hz) R2,eff is increased by ~∆R2. The reduction in Rex is eliminated when GroEL is blocked by acid-denatured Rubisco (Fig. S8). Direct binding of the folded state (F) to GroEL (i.e. exchange with a slowly tumbling species) cannot by itself lead to a reduction of Rex. To explain this observation an additional observable state (FE) that does not exchange with the unfolded state U in the presence of GroEL has to be invoked in which the folded conformation is tethered and stabilized upon confinement within the cavity of GroEL (Fig. 3). In effect, the presence of state FE reduces the population of the species that can spontaneously unfold with a concomitant decrease in Rex. The confined state FE is considered to be an observable state with the same 15N chemical shifts and comparable R2 values as those of F. ~ 22 s-1
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Figure 3. Kinetic scheme for the interaction of SH3Mut∆56 with apo GroEL. The populations of states UE and UB, displayed in grey, are below the limits of detection and were therefore not included in the fitting of the relaxation data (see SI for theory and details of fitting procedure). The rate constants and populations relate to experimental conditions of 100 µM SH3 domain and 120 µM (in subunits) GroEL at 10°C. Binding of the dimer FD to GroEL is assumed to be undetectable on account of both its size and the fact that dimerization occludes the GroEL-binding surface (Fig. S6). For SH3WT∆57 there is no evidence for the existence of an unfolded state as no relaxation dispersion is observed. The 15N-ΔR2 and DEST data for SH3WT∆57 (Fig. S7) show binding of the folded state to GroEL, and are accounted for by two-state exchange between states F and FB, with kFB and kBF values of ~7 and 500 s-1, respectively (corresponding to pB ~1.4%).
The full kinetic scheme describing all possible equilibria in the SH3Mut∆56/GroEL system is shown in Fig. 3. The folded state F is in slow exchange with the unfolded state U and the dimeric form of the protein FD in bulk solution. Observable folded (FE) and unfolded (UE) states confined within the GroEL cavity reorient rapidly; the corresponding ‘dark’, directly-bound species, FB and UB, tumble with the same rotational correlation time as GroEL. In addition, both
The complexity of the kinetic scheme in Fig. 3 precludes the definition of all the rate constants. In addition, the population (pB) and average R2 value of FB are correlated in the slow-to-intermediate exchange regime so that pB can only be defined by assuming a value of < 15 N-R2,F > consistent with the molecular weight (~800 kDa) of GroEL (~950 s-1 at 900 MHz and 10 °C; SI and Fig. S9). This enables one to define the populations of the FE (~21%) and FB (~2.6%) states, as well as the overall rate constants for the interconversion of the F and FB states through direct (F ! FB) and indirect (F ! FE ! FB) pathways; the relative contributions of these two pathways, however, cannot be determined. The overall rate constants for the conversion of F to FB and the reverse FB to F process are given by:13 B
FE
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~ 11 s-1
confined and directly-bound species can potentially undergo interconversion between folded and unfolded states. The maximal possible fraction of UE, in the absence of stabilization of FE, is
