Binding Mechanism of an SH3 Domain Studied by NMR and ITC

Abstract

Complexes between Src-homology 3 domains and proline-rich target peptides can have lifetimes on the order of milliseconds, making them too short-lived for kinetic characterization by conventional methods. Nuclear magnetic resonance (NMR) dynamics experiments are ideally suited to study such rapid binding equilibria, and additionally provide information on partly bound intermediate states. We used NMR together with isothermal titration calorimetry (ITC) to characterize the interaction of the SH3 domain from the Fyn tyrosine kinase with a 12-residue peptide at temperatures between 10 and 50 degrees C. NMR data at all temperatures are consistent with an effectively two-state binding reaction, such that any intermediates are either very weakly populated or exchange extremely rapidly with the free or bound forms. Dissociation rate constants, determined by CPMG and ZZ-exchange NMR experiments, range from k(off)(10 degrees C) = 4.5 s(-1) to k(off)(50 degrees C) = 331 s(-1). ITC data at all temperatures follow a simple two-state interaction model. Binding is favored enthalpically, with a dissociation enthalpy, DeltaH(D)(30 degrees C) = 15.4 kcal mol(-1), and disfavored entropically, with a dissociation entropy, DeltaS(D)(30 degrees C) = 20.0 cal mol(-1) K(-1). The free protein and peptide have significantly higher heat capacity than the bound complex, DeltaC(p) = 352 cal mol(-1) K(-1), which is consistent with the largely hydrophobic character of the binding interface. An Eyring plot of k(off) values gives an activation enthalpy of dissociation, DeltaH(D)(double dagger)(30 degrees C) = 19.3 kcal mol(-1) and exhibits slight curvature consistent with the ITC-derived value of DeltaC(p). The curvature suggests that nonpolar residues of the hydrophobic interface are solvated in the transition state for dissociation. Association rate constants were calculated as k(on) = k(off)/K(D), and range from k(on)(10 degrees C) = 1.03 x 10(8) M(-1) s(-1) to k(on)(50 degrees C) = 2.0 x 10(8) M(-1) s(-1), with an apparent activation enthalpy, DeltaH(A)(double dagger) = 3.4 kcal mol(-1). Both the magnitudes and temperature dependence of k(on) values are consistent with a diffusion-limited association mechanism. The combination of NMR and ITC data sheds light on how the Fyn tyrosine kinase is activated by binding to proline-rich targets, and represents a powerful approach for characterizing transient protein/ligand interactions.

Electrostatic Interactions in the Binding Pathway of a Transient Protein Complex Studied by NMR and Isothermal Titration Calorimetry

“…We previously used NMR and ITC to study the binding kinetics of the SH3 domain from the Fyn tyrosine kinase. We showed that this protein binds a type I target peptide in a two-state manner with millisecond time scale kinetics and a temperature dependence of k on that is consistent with diffusion-limited association (33). In the current study, we have focused on how positively charged residues within the SH3 binding motif and negatively charged residues in the binding site on the SH3 domain interact to influence association kinetics.…”

“…NMR-derived Binding Kinetics-We previously characterized the binding kinetics of the RR peptide using NMR and ITC over a temperature range of 10 -50°C (33). In this prior work, we employed both CPMG and ZZ magnetization exchange NMR experiments at 20°C, obtaining very similar values for k off : 11.2 and 12.8 s Ϫ1 , respectively.…”

“…The combination of nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (32) can characterize binding kinetics that are too rapid for standard stopped-flow techniques, on the order of 10 4 s Ϫ1 (33). In this approach, a substoichiometric amount of ligand is added to a protein sample whose signals are monitored by NMR spectros- copy.…”

“…The positions in the motif are numbered Ϫ3 to 3 such that the conserved ϩ occupies site Ϫ3. Dissociation constants for SH3 domains are usually on the order of micromolar, and the lifetimes of their complexes are on the order of milliseconds (33,39). We previously used NMR and ITC to study the binding kinetics of the SH3 domain from the Fyn tyrosine kinase.…”

Protein dynamics and function from solution state NMR spectroscopy

“…Global or cluster fits (assuming shared k ex values and populations) of groups of nuclei are therefore justified on physical grounds. Global fits have the advantage of increasing the precision with which exchange parameters are estimated (Demers & Mittermaier, 2009) but care must be taken with respect to how grouping is performed. Grouping can be performed by simply grouping nuclei, which yields exchange parameters similar to those obtained when nuclei are treated individually.…”