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 Crk signaling protein. Proline isomerization toggles Crk between two conformations: an autoinhibitory, 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 novel SH3 binding surfaces highlighting the binding versatility and expanding the non-canonical ligand repertoire of this important signaling domain.

NMR reveals novel mechanisms of protein activity regulation

“…62,63 The results further highlight how a 180 rotation about a single prolyl bond can have such a strong effect on the global structure of the protein. It is only in the cis conformer that the SH3 C domain exposes the specific residues that form a binding surface capable of mediating the interaction between SH3 C and SH3 N .…”

“…Proline cis-trans isomerization has emerged as a particularly efficient regulatory mechanism in many biological processes, including cell signaling, [62][63][64][65] neurodegeneration, 66 amyloidogenesis, 67 channel gating, 68 gene regulation, 69,70 phage and virus infection, 71,72 enzyme function, 73,74 and ligand recognition. 75 Proline isomerization is unique in that it exerts its function through multiple mechanisms involving (i) significant conformational changes caused by the 180 rotation about the prolyl bond, (ii) slow kinetics of isomerization affording a molecular timer, and (iii) the recruitment of prolyl cis-trans isomerase enzymes (PPIases).…”

“…Recent studies 62,63,78,79 on the Crk signaling protein have shown how proline isomerization yields a functional binary switch. Crk is an important adaptor protein consisting of a single SH2 domain, an N-terminal SH3 (SH3 N ) domain, and a C-terminal SH3 domain (SH3 C ).…”

“…We have shown that $10% of the total population of the Crk molecules exist in an open conformation wherein the PPII-binding site on SH3 is completely unoccupied. 62,63 Each Crk molecule will dynamically sample both conformations. Ligand will bind preferentially to the $10% of the Crk molecules with accessible SH3 N -binding site, as evidenced by the thermodynamic data, resulting in equilibrium shifting toward the extended, uninhibited conformation.…”

Commentary: The carboxyl‐terminal Crk SH3 domain: Regulatory strategies and new perspectives

“…Another layer of negative regulation appears to operate to prevent binding in trans to the SH3N. This is mediated via cis-trans isomerization about the Gly237-Pro238 peptide bond (in gallus CrkII) [11,12]. Intriguingly, only the cis conformer adopts an autoinhibited state whereby the hydrophobic surface of the canonical PPII binding site on the SH3N (composed of Phe142, Phe144, Trp170, Tyr187, Pro184 and Pro186) is occupied by Pro238, Phe239 and Ile270 of the SH3C in a manner similar to a PPII peptide.…”