Conformational Dynamics of Calmodulin in Complex with the Calmodulin-Dependent Kinase Kinase α Calmodulin-Binding Domain

Abstract

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...

Single-molecule force spectroscopy distinguishes target binding modes of calmodulin

“…Binding kinetics and interactions with the individual domains of CaM are largely unknown for CaMKK. However, a protein dynamics study using NMR reveals fundamentally different binding interactions of CaMKK as compared to MLCK (19). Here we show that mechanically induced peptide binding/ unbinding transitions yield detailed insights into CaM-target peptide interactions and reveal an association mode for CaMKK in which the peptide binds strongly to only partially Ca 2ϩ -saturated CaM.…”

A Surprising Role for Conformational Entropy in Protein Function

“…Calmodulin-binding domains are generally 20–30 residue sequences characterized by enrichment in basic and hydrophobic residues [88]. This system is particularly amenable to deuterium methyl relaxation experiments [32, 72, 78, 86, 89, 90]. As shown in Fig 4, methyl probes are distributed throughout the calmodulin molecule.…”

“…It is important to note that in this approach the motion of the methyl group is used to sense its local surroundings. This relies on the coupling of motion within the protein such that the “probe” methyl groups report on the local disorder [90]. This is a critical change in perspective where the dynamical probe is interpreted to reflect not only its own disorder but also the disorder of the surrounding non-probe protein matrix.…”

“…Making several simple assumptions regarding the nature of the free states so that the solvation entropy can be calculated, one can obtain in this case [90]: $$\Delta S_{\text{conf}}=mtrue[\left(n_{\text{res}}^{\text{CaM}}\mathrm{boldg}{true\langle \Delta O_{\mathrm{italicaxis}}^{2}true\rangle }^{\text{CaM}}+n_{\text{res}}^{\text{target}}\mathrm{boldg}{true\langle \Delta O_{\mathrm{italicaxis}}^{2}true\rangle }^{\text{target}}\right)true]+\Delta S_{\text{other}}$$ $$(\Delta S_{\text{tot}}-\Delta S_{\text{sol}})=mtrue[\left(n_{\text{res}}^{\text{CaM}}\mathrm{boldg}{true\langle \Delta O_{\mathrm{italicaxis}}^{2}true\rangle }^{\text{CaM}}+n_{\text{res}}^{\text{target}}\mathrm{boldg}{true\langle \Delta O_{\mathrm{italicaxis}}^{2}true\rangle }^{\text{target}}\right)true]+\Delta S_{\text{RT}}+\Delta S_{\text{other}}$$ where ΔS tot , ΔS sol , ΔS conf , ΔS RT and ΔS other are the changes in total system entropy, solvent entropy, conformational entropy, rotational-translational entropy and undocumented entropy, which is mostly solvent entropy from ion pair dissociation and solvation. The changes in side chain motion are assessed from changes in methyl order parameters weighted by the number of residues in calmodulin ($n_{\text{res}}^{\text{CaM}}$) and the target domains ($n_{\text{res}}^{\text{target}}$).…”

Application of Stopped-Flow and Time-Resolved X-Ray Absorption Spectroscopy to the Study of Metalloproteins Molecular Mechanisms

“…These protein motions were found to be critical for many biological events including enzyme catalysis, signal transduction, and protein-protein interactions [87][88][89] . Even though the three-dimensional structures of manifold functional proteins and enzymes are available, revealing molecular dynamic details that determine protein action and enzymatic functionality still represents a scientific challenge.…”