Energetics of calmodulin domain interactions with the calmodulin binding domain of CaMKII

Calmodulin (CaM) is an essential eukaryotic calcium receptor that regulates many kinases, including CaMKII. Calcium‐depleted CaM does not bind to CaMKII under physiological conditions. However, binding of (Ca2+)4‐CaM to a basic amphipathic helix in CaMKII releases auto‐inhibition of the kinase. The crystal structure of CaM bound to CaMKIIp, a peptide representing the CaM‐binding domain (CaMBD) of CaMKII, shows an antiparallel interface: the C‐domain of CaM primarily contacts the N‐terminal half of the CaMBD. The two domains of calcium‐saturated CaM are believed to play distinct roles in releasing auto‐inhibition. To investigate the underlying mechanism of activation, calcium‐dependent titrations of isolated domains of CaM binding to CaMKIIp were monitored using fluorescence anisotropy. The binding affinity of CaMKIIp for the domains of CaM increased upon saturation with calcium, with the C‐domain having a 35‐fold greater affinity than the N‐domain. Because the interdomain linker of CaM regulates calcium‐binding affinity and contribute to conformational change, the role of each CaM domain was explored further by investigating effects of CaMKIIp on site‐knockout mutants affecting the calcium‐binding sites of a single domain. Investigation of the thermodynamic linkage between saturation of individual calcium‐binding sites and CaM‐domain binding to CaMKIIp showed that calcium binding to Sites III and IV was sufficient to recapitulate the behavior of (Ca2+)4‐CaM. The magnitude of favorable interdomain cooperativity varied depending on which of the four calcium‐binding sites were mutated, emphasizing differential regulatory roles for the domains of CaM, despite the high degree of homology among the four EF‐hands of CaM. Proteins 2009. © 2008 Wiley‐Liss, Inc.

[1]  R. Kass,et al.  Calmodulin Mediates Ca2+ Sensitivity of Sodium Channels* , 2004, Journal of Biological Chemistry.

[2]  J. Lisman,et al.  The molecular basis of CaMKII function in synaptic and behavioural memory , 2002, Nature Reviews Neuroscience.

[3]  Carl A. Morris,et al.  The structure of the myosin VI motor reveals the mechanism of directionality reversal , 2005, Nature.

[4]  J. M. Bradshaw,et al.  Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II. , 2008, Biochemistry.

[5]  K. Török,et al.  Dual effect of ATP in the activation mechanism of brain Ca(2+)/calmodulin-dependent protein kinase II by Ca(2+)/calmodulin. , 2001, Biochemistry.

[6]  Acid pairs increase the N-terminal Ca2+ affinity of CaM by increasing the rate of Ca2+ association. , 2000 .

[7]  G. H. Beaven,et al.  Ultraviolet absorption spectra of proteins and amino acids. , 1952, Advances in protein chemistry.

[8]  S. Vetter,et al.  Novel aspects of calmodulin target recognition and activation. , 2003, European journal of biochemistry.

[9]  C. Bugg,et al.  Structure of calmodulin refined at 2.2 A resolution. , 1988, Journal of molecular biology.

[10]  P. Kelly,et al.  Mutagenesis of Thr-286 in monomeric Ca2+/calmodulin-dependent protein kinase II eliminates Ca2+/calmodulin-independent activity. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Hiroaki Ishida,et al.  Protein-peptide interaction studies demonstrate the versatility of calmodulin target protein binding. , 2006, Protein and peptide letters.

[12]  Jaime Prilusky,et al.  Automated analysis of interatomic contacts in proteins , 1999, Bioinform..

[13]  F A Quiocho,et al.  Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures. , 1993, Science.

[14]  F. Quiocho,et al.  Structure of calmodulin bound to the hydrophobic IQ domain of the cardiac Ca(v)1.2 calcium channel. , 2005, Structure.

[15]  R. Klevit,et al.  A series of point mutations reveal interactions between the calcium‐binding sites of calmodulin , 1992, Protein science : a publication of the Protein Society.

[16]  S. Martin,et al.  Recovery of native structure by calcium binding site mutants of calmodulin upon binding of sk-MLCK target peptides. , 1995, Biochemistry.

[17]  M. A. Shea,et al.  Quantitative endoproteinase GluC footprinting of cooperative Ca2+ binding to calmodulin: proteolytic susceptibility of E31 and E87 indicates interdomain interactions. , 1995, Biochemistry.

[18]  M. A. Shea,et al.  Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence. , 2002, Biophysical journal.

[19]  S. Smerdon,et al.  Structure of the Complex of Calmodulin with the Target Sequence of Calmodulin-Dependent Protein Kinase I: Studies of the Kinase Activation Mechanism‡ , 2002 .

[20]  A. Malmendal,et al.  Ca2+ binding and conformational changes in a calmodulin domain. , 1998, Biochemistry.

[21]  Filip Van Petegem,et al.  Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain–Ca2+/calmodulin complex , 2005, Nature Structural &Molecular Biology.

[22]  J. Johnson,et al.  Effects of Myosin Light Chain Kinase and Peptides on Ca2+ Exchange with the N- and C-terminal Ca2+ Binding Sites of Calmodulin (*) , 1996, The Journal of Biological Chemistry.

[23]  A. Nairn,et al.  A Direct Test of the Reductionist Approach to Structural Studies of Calmodulin Activity , 2002, The Journal of Biological Chemistry.

[24]  C. Klee,et al.  Ca2+ binding and conformational change in two series of point mutations to the individual Ca(2+)-binding sites of calmodulin. , 1992, The Journal of biological chemistry.

[25]  J. Hell,et al.  The NMDA receptor NR1 C1 region bound to calmodulin: structural insights into functional differences between homologous domains. , 2007, Structure.

[26]  Zenon Grabarek,et al.  Structure of a trapped intermediate of calmodulin: calcium regulation of EF-hand proteins from a new perspective. , 2005, Journal of molecular biology.

[27]  M. Waxham,et al.  A Mechanism for Calmodulin (CaM) Trapping by CaM-kinase II Defined by a Family of CaM-binding Peptides* , 1998, The Journal of Biological Chemistry.

[28]  Andy Hudmon,et al.  Neuronal CA2+/calmodulin-dependent protein kinase II: the role of structure and autoregulation in cellular function. , 2002, Annual review of biochemistry.

[29]  M. A. Shea,et al.  Interactions between domains of apo calmodulin alter calcium binding and stability. , 1998, Biochemistry.

[30]  A. Malmendal,et al.  NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state. , 1997, Biochemistry.

[31]  T. Nakatsu,et al.  Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca2+-calmodulin , 2003, Nature Structural Biology.

[32]  A. Means,et al.  Activation of four enzymes by two series of calmodulin mutants with point mutations in individual Ca2+ binding sites. , 1993, The Journal of biological chemistry.

[33]  P. Kelly,et al.  Functional analysis of a complementary DNA for the 50-kilodalton subunit of calmodulin kinase II. , 1987, Science.

[34]  Dan Wang,et al.  Comparative Analyses of the Three-dimensional Structures and Enzymatic Properties of α, β, γ, and δ Isoforms of Ca2+-Calmodulin-dependent Protein Kinase II* , 2004, Journal of Biological Chemistry.

[35]  M Ikura,et al.  Molecular and structural basis of target recognition by calmodulin. , 1995, Annual review of biophysics and biomolecular structure.

[36]  A. Persechini,et al.  Activation of myosin light chain kinase and nitric oxide synthase activities by calmodulin fragments. , 1994, The Journal of biological chemistry.

[37]  T. Craig,et al.  Restoration of the calcium binding activity of mutant calmodulins toward normal by the presence of a calmodulin binding structure. , 1991, The Journal of biological chemistry.

[38]  Hans J. Vogel,et al.  Calmodulin’s flexibility allows for promiscuity in its interactions with target proteins and peptides , 2004, Molecular biotechnology.

[39]  M. Anderson.  Calmodulin kinase signaling in heart: an intriguing candidate target for therapy of myocardial dysfunction and arrhythmias. , 2005, Pharmacology & therapeutics.

[40]  K. Beckingham,et al.  Interlobe communication in multiple calcium‐binding site mutants of Drosophila calmodulin , 1996, Protein science : a publication of the Protein Society.

[41]  D. T. Yue,et al.  Unified Mechanisms of Ca2+ Regulation across the Ca2+ Channel Family , 2003, Neuron.

[42]  D. Storm,et al.  Calcium binding to complexes of calmodulin and calmodulin binding proteins. , 1985, Biochemistry.

[43]  J. Falke,et al.  Intermolecular tuning of calmodulin by target peptides and proteins: Differential effects on Ca2+ binding and implications for kinase activation , 1997, Protein science : a publication of the Protein Society.

[44]  A. Means,et al.  Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene. , 1985, The Journal of biological chemistry.

[45]  Andy Hudmon,et al.  Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II. , 2002, The Biochemical journal.

[46]  M. A. Shea,et al.  Calcium-induced interactions of calmodulin domains revealed by quantitative thrombin footprinting of Arg37 and Arg106. , 1996, Biochemistry.

[47]  M. A. Shea,et al.  Phenylalanine fluorescence studies of calcium binding to N‐domain fragments of Paramecium calmodulin mutants show increased calcium affinity correlates with increased disorder , 2001, Protein science : a publication of the Protein Society.

[48]  M. A. Shea,et al.  Interdomain cooperativity of calmodulin bound to melittin preferentially increases calcium affinity of sites I and II , 2008, Proteins.

[49]  M. A. Shea,et al.  Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels. , 2006, Biochemistry.

[50]  J. Tainer,et al.  Structural basis for endothelial nitric oxide synthase binding to calmodulin , 2003, The EMBO journal.

[51]  J. M. Bradshaw,et al.  Thermodynamics of calmodulin trapping by Ca2+/calmodulin-dependent protein kinase II: subpicomolar Kd determined using competition titration calorimetry. , 2007, Biochemistry.

[52]  A. Bohm,et al.  Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin , 2002, Nature.

[53]  H. Vogel,et al.  Surface Exposure of the Methionine Side Chains of Calmodulin in Solution , 1999, The Journal of Biological Chemistry.

[54]  A. Edelman,et al.  Quantitation of energy coupling between Ca2+, calmodulin, skeletal muscle myosin light chain kinase, and kinase substrates. , 1984, The Journal of biological chemistry.

[55]  Richard Thorogate,et al.  Dual effect of ATP in the activation mechanism of brain Ca(2+)/calmodulin-dependent protein kinase II by Ca(2+)/calmodulin. , 2001 .

[56]  M. A. Shea,et al.  The neuronal voltage-dependent sodium channel type II IQ motif lowers the calcium affinity of the C-domain of calmodulin. , 2008, Biochemistry.

[57]  J. Johnson,et al.  Acid pairs increase the N-terminal Ca2+ affinity of CaM by increasing the rate of Ca2+ association. , 2000, Biochemistry.

[58]  K. Beckingham.  Use of site-directed mutations in the individual Ca2(+)-binding sites of calmodulin to examine Ca2(+)-induced conformational changes. , 1991, Journal of Biological Chemistry.

[59]  J. Adelman,et al.  Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin , 2001, Nature.

[60]  Guy Salama,et al.  Calmodulin kinase II inhibition protects against structural heart disease , 2005, Nature Medicine.

[61]  F A Quiocho,et al.  Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. , 1992, Science.

[62]  Stefan Mihalas,et al.  Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by calmodulin with two bound calciums , 2006, Proceedings of the National Academy of Sciences.

[63]  Anne Houdusse,et al.  Crystal structure of apo-calmodulin bound to the first two IQ motifs of myosin V reveals essential recognition features , 2006, Proceedings of the National Academy of Sciences.

[64]  M. A. Shea,et al.  An interdomain linker increases the thermostability and decreases the calcium affinity of the calmodulin N-domain. , 2002, Biochemistry.

[65]  M. Schumacher,et al.  Crystal structures of apocalmodulin and an apocalmodulin/SK potassium channel gating domain complex. , 2004, Structure.

[66]  Michael L. Johnson,et al.  [16] Nonlinear least-squares analysis , 1985 .

[67]  A. Rhoads,et al.  Sequence motifs for calmodulin recognition , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[68]  Angus C. Nairn,et al.  Structure of the Autoinhibited Kinase Domain of CaMKII and SAXS Analysis of the Holoenzyme , 2005, Cell.

[69]  D. Storm,et al.  Calcium-induced exposure of a hydrophobic surface on calmodulin. , 1980, Biochemistry.