ABSTRACT
The Src homology 3 (SH3) domain of pp60(c-src) (Src) plays dual roles in signal transduction, through stabilizing the repressed form of the Src kinase and through mediating the formation of activated signaling complexes. Transition of the Src SH3 domain between a variety of binding partners during progression through the cell cycle requires adjustment of a delicate free energy balance. Although numerous structural and functional studies of SH3 have provided an in-depth understanding of structural determinants for binding, the origins of binding energy in SH3-ligand interactions are not fully understood. Considering only the protein-ligand interface, the observed favorable change in standard enthalpy (DeltaH=-9.1 kcal/mol) and unfavorable change in standard entropy (TDeltaS=-2.7 kcal/mol) upon binding the proline-rich ligand RLP2 (RALPPLPRY) are inconsistent with the predominantly hydrophobic interaction surface. To investigate possible origins of ligand binding energy, backbone dynamics of free and RLP2-bound SH3 were performed via (15)N NMR relaxation and hydrogen-deuterium (H/(2)H) exchange measurements. On the ps-ns time scale, assuming uncorrelated motions, ligand binding results in a significant reduction in backbone entropy (-1.5(+/-0.6) kcal/mol). Binding also suppresses motions on the micros-ms time scale, which may additionally contribute to an unfavorable change in entropy. A large increase in protection from H/(2)H exchange is observed upon ligand binding, providing evidence for entropy loss due to motions on longer time scales, and supporting the notion that stabilization of pre-existing conformations within a native state ensemble is a fundamental paradigm for ligand binding. Observed changes in motion on all three time scales occur at locations both near and remote from the protein-ligand interface. The propagation of ligand binding interactions across the SH3 domain has potential consequences in target selection through altering both free energy and geometry in intact Src, and suggests that looking beyond the protein-ligand interface is essential in understanding ligand binding energetics.
Subject(s)
Proto-Oncogene Proteins pp60(c-src)/chemistry , Proto-Oncogene Proteins pp60(c-src)/metabolism , src Homology Domains , Animals , Anisotropy , Binding Sites , Calorimetry , Chickens , Deuterium/metabolism , Hydrogen Bonding , Kinetics , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Motion , Nitrogen Isotopes , Protein Binding , Protein Denaturation , Protein Folding , ThermodynamicsABSTRACT
Although an accurate description of global tumbling of a protein is essential for correct analysis of internal motions. proper distinction between the effects of anisotropic rotational diffusion and conformational exchange has remained a challenge. We present a novel two-part filtering procedure designed specifically to distinguish between the effects of anisotropy and conformational exchange. The efficacy of this method is assessed using synthetic data sets. The method is then applied to two proteins of dramatically different size and shape, OspA and SH3. The large size and extreme anisotropy of OspA provide a challenging case, where conformational exchange is a small perturbation of the effects of anisotropy on transverse relaxation rates. Conversely, in the chicken c-Src SH3 domain, with its small size and nearly spherical shape, anisotropy is a small perturbation of the effects of conformational exchange on transverse relaxation rates. Accurate extraction of the global tumbling parameters for each protein allows optimal characterization of conformational exchange processes, as well as ps-ns time scale motions.
Subject(s)
Lipoproteins , Nuclear Magnetic Resonance, Biomolecular/methods , Proteins/chemistry , Animals , Anisotropy , Antigens, Surface/chemistry , Bacterial Outer Membrane Proteins/chemistry , Bacterial Vaccines , Crystallography, X-Ray , Models, Molecular , Nitrogen Isotopes , Protein Conformation , src Homology DomainsABSTRACT
We studied the effect of pyridostigmine bromide, a nerve agent prophylactic, on the central nervous system (CNS) uptake of [14C]permethrin, a pyrethroid insecticide, at scaled human-equivalent exposures in rats using accelerator mass spectrometry (AMS). AMS detects 14C at attomole sensitivities and determines the tissue distribution of 14C-labeled compounds. Pyridostigmine bromide in chow at 7.75 mg kg(-1) per day lowered the CNS tissue levels of permethrin, dosed at 4.75 microg kg(-1), in the CNS of rats by 30%. These results are inconsistent with hypothesized synergy of such compounds as a precursor to 'Gulf War syndrome'.
