title: Thermodynamic dissection of the Ezrin FERM/CERMAD interface.
abstract: ERM (Ezrin-Radixin-Moesin) proteins are key cross-linkers of the plasma membrane and the actin cytoskeleton. They are regulated by the intramolecular association of the N-terminal FERM (band-four point one, Ezrin, Radixin, Moesin) and C-terminal CERMAD (ERM association domain) domains (N/C interaction), which masks the binding surfaces of the domains for other molecules. The N/C interface is characterized by the highly distributed binding of CERMAD through a beta-strand and four alpha-helices to a globular FERM. Though it is a target for multiple regulatory signals, little is known about the dynamics/thermodynamics governing this interface. Recent implications of Ezrin in cancer metastasis have increased the necessity to understand this regulatory switch. In this study, we report residue-specific stabilities of Ezrin CERMAD at the Ezrin N/C interface obtained using hydrogen-deuterium exchange NMR. These stabilities vary across secondary structural elements and identify F583 and L586 as key anchor residues for the most stable element, alphaD. Macroscopic N/C binding energetics, obtained using isothermal titration calorimetry (ITC) reveals a high affinity (Kd =176 nM) enthalpy-driven binding (DeltaH = -26 kcal/mol, TDeltaS = -17 kcal/mol) at 25 degrees C at pH 7 in MES and phosphate buffers. A 10-fold increase in affinity was observed for measurements in acetate buffer, suggesting that an acetate-like molecule might promote the repressed form of the complex, possibly through interaction with the F2 subdomain of FERM, which resembles the acyl-CoA binding protein. In summary, our results have illustrated the dynamic nature of this regulatory interface and provide a foundation for investigating the role of regulatory signals on the stability of this interface.
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Bhargavi
authors.initials: B
authors.lastname: Jayaraman
authors.forename: Linda K
authors.initials: LK
authors.lastname: Nicholson
mesh_terms: D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D006859:Hydrogen; D000099149:Ezrin