Structural basis for regulation of the Crk signaling protein by a proline switch

Abstract

Proline switches, controlled by cis-trans isomerization, have emerged as a particularly effective regulatory mechanism in a wide range of biological processes. Here we report the structures of both the cis and trans conformers of a proline switch in the Crk signaling protein. Proline isomerization toggles Crk between two conformations: an autoinhibitory conformation, stabilized by the intramolecular association of two tandem SH3 domains in the cis form, and an uninhibited, activated conformation promoted by the trans form. In addition to acting as a structural switch, the heterogeneous proline recruits cyclophilin A, which accelerates the interconversion rate between the isomers, thereby regulating the kinetics of Crk activation. The data provide atomic insight into the mechanisms that underpin the functionality of this binary switch and elucidate its remarkable efficiency. The results also reveal new SH3 binding surfaces, highlighting the binding versatility and expanding the noncanonical ligand repertoire of this important signaling domain.

Figure 1. Domain organization of Crk

(a) Schematic diagram of the domain organization of Crk. Pro238, which undergoes cis–trans isomerization, and the tyrosine residue (Tyr222) that becomes phosphorylated by Abl are indicated. (b) Schematic of cis–trans isomerization about the prolyl Gly237–Pro238 bond.

Figure 2. Structural characterization of the trans and cis Crk l-SH3^C conformers

(a,b) Structure of the Crk l-SH3^C polypeptide in the trans conformation and (c,d) in the cis conformation. In (a and c) the SH3^C domain is colored light blue and the linker is colored grey. Pro238 is shown in red. N and C denote the N- and C-terminus. In (b and d) the SH3^C domain is displayed as a semi-transparent surface and residues involved in the linker–SH3^C interactions are highlighted. Linker and SH3^C residues are colored green and yellow, respectively. Dotted lines denote hydrogen bonding.

Figure 3. Structural comparison of the trans and cis isomers

(a) Superposition of the trans and cis conformers of l-SH3^C on the SH3^C domain (residues 239–295). The SH3^C domain in the trans and cis conformation is in light and dark grey, respectively. The linker adopts a very different structure in the trans (green) and the cis (purple) conformation. Representative residues at the linker are indicated. The conformation of the n-src loop is distinct in the trans (green) and the cis (purple) conformer. (b) Effect of cis–trans isomerization on the structure of SH3^C. The side-chain conformation of select residues is shown. The side chain of Phe239 is solvent exposed in the cis isomer (purple) whereas it is buried in the trans isomer (green). Superposition of the structures and color code are as in (a). (c) Leu231 is completely buried into a hydrophobic pocket in SH3^C in the cis conformation. The side-chain conformation of residues lining the hydrophobic pocket is shown in the cis (purple) and trans (green) isomers.(d,e) Solvent-accessible surface presentation of the trans (d) and cis (e) conformations. The models are rotated ~60° about the z axis relative to the models in (a) with the linker facing the reader. The surface presented by the linker is delineated.

Figure 4. Dynamic characterization of the trans and cis isomers

(a) N-H bond order parameters, S², plotted as a function of the primary sequence, for the trans (blue) and cis (red) conformers of l-SH3^C. (b) Changes in order parameters, ΔS², between the trans and the cis conformers mapped using a gradient color code on the structure of cis l-SH3^C. ΔS² is given as S² (trans) – S² (cis), so positive ΔS² values denote enhanced rigidity of the protein backbone in the trans over the cis conformation.

Figure 5. Structural characterization of Crk^SLS in the closed, autoinhibited conformation

(a) Lowest-energy structure of Crk^SLS shown as cartoon with SH3^N in pink, SH3^C in light blue, and linker in grey. (b) Close-up view of the SH3^N–SH3^C interface. SH3^N and SH3^C residues are colored green and orange, respectively. (c) View from above SH3^C, which is shown as transparent blue cartoon. SH3^N is displayed as pink solvent-accessible surface. SH3^C residues interacting with SH3^N are shown as orange sticks. (d) Structure of SH3^N in complex with a consensus PPII peptide (P-x-P-L-x-K). The orientation of SH3^N in (c) and (d) is the same. Comparison of (c) and (d) show that the PPII-binding site in Crk^SLS is not accessible.

Figure 6. Structural basis for conformer-specific SH3^N–SH3^C interaction in Crk^SLS

(a) Superposition of the cis and trans l-SH3^C and Crk^SLS on the SH3^C domain. The side chains of Pro238, Phe239, and Ile270, residues primarily mediating the SH3^N–SH3^C interaction, are shown in green for trans and yellow for cis l-SH3^C and orange for Crk^SLS. SH3^N is shown as solvent-accessible surface. (b) Effect of single amino acid substitution on the stability of the closed conformation of Crk^SLS as assessed by measuring the population of the closed and open conformations of Crk^SLS by NMR. ΔG for P238A and L231G is a lower-bound limit since populations less than ~5% are beyond the NMR detection limit.

Figure 7. Mechanistic basis for the the regulation of Crk activity

Crk adopts predominantly (~90%) the closed, auto-inhibited conformation but a minor population (~10%) adopts the open, uninhibited conformation wherein the PPII-binding site on SH3^N is accessible for binding. The P-x-L-P-x-K motif of Abl binds to SH3^N with a relatively strong affinity (K_d ~1 μM). The open conformation exists as an equilibrium between the cis and the trans isomer, but only the cis one forms the closed conformation. The rates of the cis–trans interconversion are regulated by the action of CypA, which accelerates the interconversion by four orders of magnitude.