Ultrasonic and densimetric characterization of the association of cyclic AMP with the cAMP-binding domain of the exchange protein EPAC1

We employed a combination of densimetric and ultrasonic velocimetric techniques to characterize the volumetric properties of the association of the cAMP-binding domain (CBD) of EPAC1 with cAMP at 25 C in a pH 7.6 buffer. The binding of cAMP to the CBD of EPAC1 is accompanied by changes in volume, ΔV, and adiabatic compressibility, ΔK_S, of -59 ± 4 cm³ mol^-1 and (34 ± 9) × 10^-4 cm³ mol^-1 bar^-1, respectively. We use these volumetric results in conjunction with the structural data to estimate a change in hydration, Δn_h, accompanying the binding. We calculate that approximately 103 water molecules are released to the bulk from the associating surfaces of the protein and the ligand. This number is ∼30% larger than the number of water molecules in direct contact with the associating surfaces while also being within the error of our Δn_h determination. Therefore, we conclude that cAMP binding to EPAC1 may involve, in addition to the waters from within the first coordination sphere, also some waters from the second coordination sphere of the protein and cAMP. Our analysis of the compressibility data reveals that the protein becomes more rigid and less dynamic upon the cAMP binding as reflected in a 4 ± 0.5% decrease in its intrinsic coefficient of adiabatic compressibility. Finally, we estimate the hydration, ΔS_hyd, and configurational, ΔS_conf, contributions to the binding entropy, ΔS_b. We find that the binding entropy is determined by the fine balance between the ΔS _hyd and ΔS_conf terms. In general, we discuss insights that are derived from a combination of volumetric and structural properties, in particular, emphasizing how measured changes in volume and compressibility can be interpreted in terms of hydration and dynamic properties of EPAC1 in its apo- and holo-forms.