Kinetic Insights into the Binding between the nSH3 Domain of CrkII and Proline-Rich Motifs in cAbl

Abstract

The interaction between CrkII and cAbl is implicated in diverse cellular processes. This interaction starts with the binding of the N-terminal Src homology 3 (nSH3) domain of CrkII to the proline-rich motifs of cAbl (PRMs). Despite its critical importance, the detailed binding mechanism between the nSH3 domain and PRMs remains elusive. In this study, we used nuclear magnetic resonance Carr-Purcell-Meiboom-Gill relaxation dispersion experiment to study the binding kinetics between the nSH3 domain of CrkII and PRMs. Our results highlight that the nSH3 domain binds to three PRMs with very high on- and off-rate constants, indicating the transient nature of the binding. To further characterize the binding transition state, we conducted the Eyring and linear free energy relationship analyses using temperature-dependent kinetic data. These data indicate that the binding transition state of the nSH3 domain and PRM is accompanied by small activation enthalpy, owing to partial desolvation of the transition state. These results also highlight the similarity between the transition and free states, in terms of structure and energetics. Although the binding of the nSH3 domain and PRM displays the features consistent with a diffusion-limited process within our experimental conditions, further tests are necessary to determine if the binding is a true diffusion-limited process.

Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis

“…The kinetic and equilibrium results from 1D 19 F lineshape analysis are consistent with other reports on SH3-peptide interactions (62,(79)(80)(81). The kinetic investigations (62,81) found that dissociation was more sensitive to temperature change than association, and the signs of the energetic terms were the same.…”

“…The kinetic and equilibrium results from 1D 19 F lineshape analysis are consistent with other reports on SH3-peptide interactions (62,(79)(80)(81). The kinetic investigations (62,81) found that dissociation was more sensitive to temperature change than association, and the signs of the energetic terms were the same. Importantly, other reports used alternative techniques including isothermal titration calorimetry (80,81), fluorescence spectroscopy (62,79), ZZ-exchange NMR (81), and Carr-Purcell-Meiboom-Gill relaxation dispersion NMR (62,81).…”

“…The favorable TDS 0 z D is likely driven by the increase in conformational freedom of the protein and peptide upon dissociation. The signs and magnitudes of all kinetic and equilibrium thermodynamic parameters agree with other studies of SH3 interactions with proline-rich peptides (62,79,81).…”

“…S7). Others report similar trends for a different SH3-peptide interaction (62). We conclude that the temperatures used here minimally perturb the structure of free and bound SH3, which simplifies interpretation of the temperature dependence of peptide binding presented later.…”

“…The kinetic investigations (62,81) found that dissociation was more sensitive to temperature change than association, and the signs of the energetic terms were the same. Importantly, other reports used alternative techniques including isothermal titration calorimetry (80,81), fluorescence spectroscopy (62,79), ZZ-exchange NMR (81), and Carr-Purcell-Meiboom-Gill relaxation dispersion NMR (62,81). The broad agreement of our data with those obtained with other biophysical techniques shows that 1D 19 F lineshape analysis is robust.…”