Abstract
c-CrkII is a central signal adapter protein. A domain opening/closing reaction between its N- and C-terminal Src homology 3 domains (SH3N and SH3C, respectively) controls signal propagation from upstream tyrosine kinases to downstream targets. In chicken but not in human c-CrkII, opening/closing is coupled with cis/trans isomerization at Pro-238 in SH3C. Here, we used advanced double-mixing experiments and kinetic simulations to uncover dynamic domain interactions in c-CrkII and to elucidate how they are linked with cis/trans isomerization and how this regulates substrate binding to SH3N. Pro-238 trans → cis isomerization is not a simple on/off switch but converts chicken c-CrkII from a high affinity to a low affinity form. We present a double-box model that describes c-CrkII as an allosteric system consisting of an open, high affinity R state and a closed, low affinity T state. Coupling of the T-R transition with an intrinsically slow prolyl isomerization provides c-CrkII with a kinetic memory and possibly functions as a molecular attenuator during signal transduction.
Highlights
Signaling by chicken c-CrkII involves native state prolyl isomerization
We employed advanced kinetic techniques, in particular double-mixing experiments, to uncover domain interactions in c-CrkII and to elucidate how they are linked energetically with Pro-238 cis/trans isomerization in SH3C and with substrate binding to the SH3 domain (SH3N) domain. Based on these data and on kinetic simulations, we present a detailed mechanism that reveals the role of prolyl isomerization for the folding and the function of chicken c-CrkII, and we propose that the intrinsically slow isomerization at Pro-238 functions as a molecular memory, possibly as an attenuator, during the allosteric regulation of c-CrkII signaling
In the chicken but not in the human protein, the opening/closing reaction is coupled with cis/trans isomerization at Pro-238 in the SH3C domain
Summary
Signaling by chicken c-CrkII (cellular CT10 regulator of kinase) involves native state prolyl isomerization. We employed advanced kinetic techniques, in particular double-mixing experiments, to uncover domain interactions in c-CrkII and to elucidate how they are linked energetically with Pro-238 cis/trans isomerization in SH3C and with substrate binding to the SH3N domain. Based on these data and on kinetic simulations, we present a detailed mechanism that reveals the role of prolyl isomerization for the folding and the function of chicken c-CrkII, and we propose that the intrinsically slow isomerization at Pro-238 functions as a molecular memory, possibly as an attenuator, during the allosteric regulation of c-CrkII signaling
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