Tuning protein autoinhibition by domain destabilization

Journal name:
Nature Structural & Molecular Biology
Year published:
Published online


Activation of many multidomain signaling proteins requires rearrangement of autoinhibitory interdomain interactions that occlude activator binding sites. In one model for activation, the major inactive conformation exists in equilibrium with activated-like conformations that can be stabilized by ligand binding or post-translational modifications. We established the molecular basis for this model for the archetypal signaling adaptor protein Crk-II by measuring the thermodynamics and kinetics of the equilibrium between autoinhibited and activated-like states. We used fluorescence and NMR spectroscopies together with segmental isotopic labeling by means of expressed protein ligation. The results demonstrate that intramolecular domain-domain interactions both stabilize the autoinhibited state and induce the activated-like conformation. A combination of favorable interdomain interactions and unfavorable intradomain structural changes fine-tunes the population of the activated-like conformation and allows facile response to activators. This mechanism suggests a general strategy for optimization of autoinhibitory interactions of multidomain proteins.

At a glance


  1. Scheme for the autoinhibition and activation of Crk-II.
    Figure 1: Scheme for the autoinhibition and activation of Crk-II.

    (a) Solution structure of Crk-II (SH2, blue; nSH3, orange; cSH3, green) (PDB 2EYZ19). Active-site residues of each domain are shown in stick-and-ball format. (b) Expanded view of the interface between the active site of the nSH3 and SH2 domains in Crk-II. Active-site residues are shown in red on the surface representation of the nSH3 domain. Interface residues of the SH2 domain are shown in stick format. (c) Schematic representation of a three-state equilibrium of ligand binding to the nSH3 domain in Crk-II (SH2, blue; nSH3, orange; cSH3, green; ligand, red). KO is the equilibrium constant between the closed and open state, and Kint is the intrinsic binding affinity of the ligand to the nSH3 domain in the open state. C and O represent the closed and open state of Crk-II, respectively. L represents the ligand that binds to the nSH3 domain. (d) Change in the chemical shift of Trp169 (top) and Ala172 (bottom) as a function of C3G-ligand concentration. The ratios of protein to ligand are 1:0 (red), 1:0.22 (green), 1:0.63 (cyan), 1:0.79 (black) and 1:1.19 (blue).

  2. NMR analysis of the cSH3 domain within full-length Crk-II.
    Figure 2: NMR analysis of the cSH3 domain within full-length Crk-II.

    (a) Schematic diagram outlining preparation of segmentally labeled Crk-II (residues 208–304 are labeled with 15N). COSR represents the C-terminal α-thioester derivative. (b) Overlay of the 1H-15N HSQC spectrum of uniformly 15N-labeled Crk-II (black) and segmentally labeled Crk-II (red). (c) NMR-detected H/D-exchange free energy of unfolding of the cSH3 domain in isolation (black) and Crk-II (gray). The error bars represent s.d. of three repeated experiments. (d) Residues detected in the H/D-exchange experiment are shown as spheres on the structure of the cSH3 domain.

  3. Structural and dynamic analysis of the cSH3 domain within Crk-II.
    Figure 3: Structural and dynamic analysis of the cSH3 domain within Crk-II.

    (a) Chemical-shift differences (Δδ) of the cSH3 domain in Crk-II (black) and C3G ligand–bound Crk-II (green), relative to the isolated cSH3 domain. The inset shows the locations of the residues whose resonances changed markedly between isolated cSH3 and Crk-II (top 25% in the deviation plot). (b) Chemical-shift differences of the cSH3 domain in C3G ligand–bound Crk-II compared to those within Crk-II. The difference for the 15Nɛ1 resonance of Trp275 is shown as a bar. (c) Comparison of average R2 rates of the cSH3 domain in isolation (molecular mass = 8,569 Da), in Crk-II, and in Crk-II and C3G (~33,830 Da). The dashed line represents the molecular mass–dependent R2 rates calculated using isotropic rotational correlation times from Stokes' law. Error bars represent the s.d. of average R2. (d) {1H}-15N heteronuclear NOE measurements for the cSH3 domain in isolation, residues 232–304 (closed circles), and within Crk-II, residues 208–304 (open circles). The residues corresponding to the linker region, residues 208–236, are shaded in the plot. Error bars represent the propagated uncertainties of two repeated experiments. The background noise of the spectrum was used to estimate the uncertainty.

  4. Conformation of Trp275 modulates the stability of the cSH3 domain.
    Figure 4: Conformation of Trp275 modulates the stability of the cSH3 domain.

    (a) Comparison of the position of the indole ring of Trp275 in the structures of isolated cSH3 domain (green, PDB 2GGR32) and full-length Crk-II (silver, PDB 2EYZ19). The 7-position of the tryptophan residue is highlighted in red. (b) Steady-state fluorescence quenching experiments for free indole (closed circles) and 7-azaindole (open circles). (c) Steady-state fluorescence quenching experiments for isolated cSH3-WT (closed circles) and isolated cSH3-7AW (open circles). The solid lines represent the best-fit model using the Stern-Volmer relationship (see Online Methods). (d) Schematic representation of the effects of the conformational change of Trp275 on the equilibrium between the open and closed states of Crk-II (red 'W' represents Trp275). Reduced stability of the cSH3 upon interdomain interactions reduces the activation barrier between the open and closed states of Crk-II.


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Author information


  1. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA.

    • Jae-Hyun Cho &
    • Arthur G Palmer III
  2. The Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, New York, USA.

    • Vasant Muralidharan,
    • Miquel Vila-Perello &
    • Tom W Muir
  3. Department of Chemistry and Graduate Program in Biochemistry and Structural Biology, State University of New York at Stony Brook, Stony Brook, New York, USA.

    • Daniel P Raleigh
  4. Present address: Howard Hughes Medical Institute, Washington University School of Medicine, Departments of Molecular Microbiology and Medicine, St. Louis, Missouri, USA.

    • Vasant Muralidharan


J.-H.C. designed and conducted all experiments, analyzed the data and helped write the paper. V.M. and M.V.-P. helped to synthesize the ligands and prepare the segmentally labeled protein. D.P.R., T.W.M. and A.G.P. designed the experiments, analyzed the data and helped write the paper.

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The authors declare no competing financial interests.

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