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.

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 ).…”

“…It is of particular interest that cyclophilin A (CypA) is recruited and accelerates dramatically the rate of the interconversion to the millisecond regime at physiological temperatures, a more meaningful timescale for modulating the kinetics of biological processes. 62 Thus, proline isomerization in Crk functions as a molecular timer whereby the energetics and rate of signaling complex formation can be regulated.…”

“…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.…”