Molecular recognition by proteins is fundamental to almost every biological process, particularly the protein associations underlying cellular signal transduction. Understanding the basis for protein-protein interactions requires the full characterization of the thermodynamics of their association. Historically it has been virtually impossible to experimentally estimate changes in protein conformational entropy, a potentially important component of the free energy of protein association. However, nuclear magnetic resonance spectroscopy has emerged as a powerful tool for characterizing the dynamics of proteins. Here we employ changes in conformational dynamics as a proxy for corresponding changes in conformational entropy. We find that the change in internal dynamics of the protein calmodulin varies significantly on binding a variety of target domains. Surprisingly, the apparent change in the corresponding conformational entropy is linearly related to the change in the overall binding entropy. This indicates that changes in protein conformational entropy can contribute significantly to the free energy of protein-ligand association.

The physical basis for high affinity interactions involving proteins is complex and potentially involves a range of energetic contributions. Among these are changes in protein conformational entropy, which cannot yet be reliably computed from molecular structures. We have recently employed changes in conformational dynamics as a proxy for changes in conformational entropy of calmodulin upon association with domains from regulated proteins. The apparent change in conformational entropy was linearly related to the overall binding entropy. This view warrants a more quantitative foundation. Here we calibrate an “entropy meter” employing an experimental dynamical proxy based on NMR relaxation and show that changes in the conformational entropy of calmodulin are a significant component of the energetics of binding. Furthermore, the distribution of motion at the interface between the target domain and calmodulin are surprisingly non-complementary. These observations promote modification of our understanding of the energetics of protein-ligand interactions.

As the primary intracellular calcium sensor, calmodulin (CaM) regulates numerous and diverse proteins. Several mechanisms, including tissue specific expression, localization and sequestration, work in concert to limit the total number of available targets of calmodulin within a cell. While the free energies of binding of calmodulin-binding domains of regulated proteins by CaM have been shown to be highly similar, they result from vastly different enthalpic and entropic contributions. Here, we report the backbone and side-chain methyl dynamics of calcium-activated calmodulin in complex with a peptide corresponding to the CaM binding domain of calmodulin kinase kinase, along with the thermodynamic underpinnings of complex formation. The results show a considerable reduction in side-chain mobility throughout CaM upon binding the CaMKKα peptide which is consistent with the enthalpically driven nature of the binding. Site specific comparison to another kinase-derived peptide complex with similar thermodynamic values, reveals significant differences in dynamics largely localized to the hydrophobic binding sites.Calmodulin is a highly conserved protein, capable of binding and regulating numerous proteins in response to increased levels of intracellular calcium (1,2). Calcium-saturated calmodulin (CaM) is the dominant regulatory species though there are several cases where apocalmodulin is found to bind target proteins. Current estimates indicate well over 300 proteins contain a CaM binding motif (3). Critical among these are kinases which subsequently regulate gene transcription, muscle contraction, transport, and other cellular processes (4). Calcium/ calmodulin-dependent protein kinase kinase (CaMKK) acts to enhance the activity of downstream CaM-dependent kinases (types I and IV) by phosphorylation of a single Thr in the so-called activation loop (5-7). CaMKI has recently been shown to function within the ERK signaling pathway (8) and CaMKI and CaMKIV differentially activate the transcription factors CREB, CREM, and ATF-1 (9-11). Two isoforms of CaMKK are known (α and β) each with abundant expression in neuronal, although CaMKKβ also has low level expression in the † Supported by NIH research grant DK 39806. *To whom correspondence should be addressed. Email: wand@mail.med.upenn.edu. Contact Information: Professor A. Joshua Wand, Department of Biochemistry & Biophysics University of Pennsylvania Philadelphia, PA 19104-6059, Tel: 215-573-7288, Fax: 215-573-7290, E-mail: wand@mail.med.upenn.edu 1 Abbreviations used: CaM, calcium-saturated calmodulin; CaMKK, calcium/calmodulin-dependent protein kinase kinase; CaMKKαp, peptide based on the CaMKKα calmodulin-binding domain with sequence CaMKKα sequence: GSVKLIPSWTTVILVKSMLRKRSFGNPF; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; NOE, { 1 H}-15 N nuclear Overhauser effect; and , Lipari-Szabo squared generalized order parameters for the methyl group symmetry axis and amide N-H bond, respectively; PCR, polymerase chain reaction; rmsd, root m...