Proline cis-trans isomerization controls autoinhibition of a signaling protein

Abstract

Autoinhibition is being widely used in nature to repress otherwise constitutive protein activities and is typically regulated by extrinsic factors. Here we show that autoinhibition can be controlled by an intrinsic intramolecular switch afforded by prolyl cis-trans isomerization. We find that a proline on the linker tethering the two SH3 domains of the Crk adaptor protein interconverts between the cis and trans conformation. In the cis conformation, the two SH3 domains interact intramolecularly, thereby forming the basis of an autoinhibitory mechanism. Conversely, in the trans conformation Crk exists in an extended, uninhibited conformation that is marginally populated but serves to activate the protein upon ligand binding. Interconversion between the cis and trans, and, hence, of the autoinhibited and activated conformations, is accelerated by the action of peptidyl-prolyl isomerases. Proline isomerization appears to make an ideal switch that can regulate the kinetics of activation, thereby modulating the dynamics of signal response.

Figure 1. Proline isomerization induces conformational heterogeneity within the SH3^C domain and is catalyzed by CypA

(A) Schematic diagram of the domain organization of Crk. The various Crk fragments used in this study are indicated. Cross peaks of spectra in all figures are colored according to the indicated color code. Highly conserved proline linker residues are indicated in bold. Pro238, which undergoes cis-trans isomerization, is highlighted. The tyrosine residue (Y222) that becomes phosphorylated by Abl is also indicated. (B) ¹H-¹⁵N HSQC of hl-SH3^C. Representative assignment of both cis (c) and trans (t) conformations is included. (C) Characteristic NOE cross peaks from ¹³C-edited NOESY spectra between the Gly237-Hα₁ and Pro238-Hα protons for the cis conformation, and the Gly237-Hα₁ and Pro238-Hδ_1,2 protons for the trans conformation. (D) Strips from CCO-NH spectra showing the ¹³C chemical shift of Pro238 carbon skeleton. The chemical shift difference between the ¹³C_β and the ¹³C_γ nuclei for the two conformations of residue Pro238 are 9.2 and 4.6 ppm, which, on the basis of a statistical analysis of ¹³C chemical shifts of proline residues in proteins (Schubert et al., 2002), further corroborates that the two forms correspond to the cis and trans conformations of the Gly237–Pro238 bond. (E) Effect of cis-trans isomerization at Pro238 on SH3^C, assessed by chemical shift mapping. Chemical shift difference (Δδ) between the two conformations is mapped by continuous-scale color onto the structure of SH3^C (PDB entry 2GGR). Proline residues are colored white. Numerical values of Δδ are given in Figure S2. (F) 2D ¹H-¹⁵N heteronuclear (ZZ) NMR exchange spectra of fl-SH3^C in the absence and presence of catalytic amounts of CypA. Exchange peaks, indicated within the dotted lines, appear when the rate of interconversion between the cis and trans conformations is relatively fast. The determined uncatalyzed and catalyzed rates of interconversion are included.

Figure 2. Proline Cis -transisomerization regulates the linker -SH3^Cinteraction

(A) Overlaid ¹H-¹⁵N HSQC spectra of isolated SH3^C (magenta) and hl-SH3^C (blue). (B) Chemical shift difference (Δδ) between isolated SH3^C and the cis (blue) and trans (red) conformers of hl-SH3^C plotted as a bar graph and (C) mapped (for the trans conformation) using a continuous-scale color on the structure of SH3^C. In (B) the (o) symbols indicate Pro residues.

Figure 3. Intramolecular interaction between the SH3^N and SH3^C domains

(A) Overlaid ¹H-¹⁵N HSQC spectra of isolated SH3^N (cyan), fl-SH3^C (green) and SH3^N-fl-SH3^C (red). Representative assignment exemplifying the large chemical shift change upon intramolecular interaction for the SH3^N (cyan) and SH3^C (magenta), as well as the selection of only the cis conformer upon binding is indicated. (B) Chemical shift difference (Δδ) between SH3^N-fl-SH3^C and its isolated components SH3^N and fl-SH3^C plotted as a bar graph and (C) mapped using a continuous-scale color on the structures of SH3^N and SH3^C. In (B) the (o) symbols indicate Pro residues. In (C) SH3^N is displayed with a P-x-L-P-x-K peptide bound (brown sticks; PDB entry 1CKA) to simply indicate the PPII binding site.

Figure 4. The PPII ligand and SH3^C binding sites on SH3^N are mutually exclusive resulting in inhibition ofSH3^N-mediated complex formation

(A) Overlaid ¹H-¹⁵N HSQC spectra of isolated SH3^N in complex with the PPII peptide (cyan), fl-SH3^C (green) and SH3^N-fl-SH3^C in complex with the PPII peptide (red). (B) Binding isotherms of the calorimetric titration of PPII peptide to isolated SH3^N (cyan) and SH3^N-fl-SH3^C (red). (C) Thermodynamic parameters of the titration experiments described in (B). (D) Western blots of lysates from 293T cells transfected with Abl plus wild-type Crk and mutants. In the top panel, immune complexes were electrophoretically resolved and probed with anti-Abl antibody (to witness Abl co-immunoprecipitation). In the lower panel, the immunoblot was re-probed with anti-Crk to show that equivalent Crk was imuunoprecipitated in each lane. The F239A mutation, which disrupts the autoinhibitory conformation, increases significantly the Crk-Abl association.

Figure 5. TheF239A mutation disrupts the autoinhibitory conformation

(A) Effect of the F239A mutation on the autoinhibitory conformation of SH3^N-fl-SH3^C. Overlaid ¹H-¹⁵N HSQC spectra of SH3^N-fl-SH3^C (red), SH3^N-fl-SH3^C-F239A (orange) and isolated SH3^N (cyan). F239A mutation results in all resonances of the SH3^N domain in SH3^N-fl-SH3^C shifting to the corresponding chemical shifts of the isolated domain (characteristic shifts are indicated in the figure). Therefore, this mutation abolishes the autoinhibitory conformation. (B) Intramolecular interaction between SH3^N and SH3^C. Red-coloured regions are the ones mostly affected by the interaction based on Δδ. The highly conserved aromatic residues that most likely mediate the binding are highlighted. The PPII peptide is shown bound to SH3^N to simply indicate the binding site.

Figure 6. Equilibrium of autoinhibited (“closed”) and uninhibited (“open”) conformation alstates controlled by proline cis-transisomerization

(A) Overlaid ¹H-¹⁵N HSQC spectra of isolated SH3^N (cyan), fl-SH3^C (green) and SH3^N-fl-SH3^C (red) indicating the presence of a minor conformation of SH3^N-fl-SH3^C in an “open”, uninhibited conformation. In this conformation the SH3^N binding site is completely accessible and the Gly237-Pro238 prolyl bond adopts only the trans conformation. SH3^N residues in the minor conformation are primed. (B) Ratio of T₁ over T₂ relaxation rates of the major (red) and minor (blue) conformation of Crk SH3^N-fl-SH3^C polypeptide. The major and minor conformations correspond to a “closed” and “open” intramolecular conformation, respectively. The points for the minor conformation are much fewer than for the major conformation because of the lower intensity of the minor cross peaks. The T₁/T₂ ratio provides information about the correlation time(tumbling) of the molecule. Higher values indicate slower tumbling.

Figure 7. Model of the equilibrium of conformational states of Crk SH3^N-fl-SH3^C polypeptide, its autoinhibition and activation

The intramolecular inhibitory SH3^N/SH3^C interaction is stabilized by the cis conformer of the Gly237-Pro238 prolyl bond, whereas the trans conformer favors an uninhibited state. Activation occurs by PPII ligand binding to a low population of uninhibited states wherein SH3^N binding site is accessible, thereby shifting the equilibrium towards the SH3^N-PPII ligand bound state. Pro238 acts as a molecular switch that has the intrinsic capacity to regulate the autoinhibition of Crk.