Structural Analysis and Stochastic Modelling Suggest a Mechanism for Calmodulin Trapping by CaMKII

Activation of CaMKII by calmodulin and the subsequent maintenance of constitutive activity through autophosphorylation at threonine residue 286 (Thr286) are thought to play a major role in synaptic plasticity. One of the effects of autophosphorylation at Thr286 is to increase the apparent affinity of CaMKII for calmodulin, a phenomenon known as “calmodulin trapping”. It has previously been suggested that two binding sites for calmodulin exist on CaMKII, with high and low affinities, respectively. We built structural models of calmodulin bound to both of these sites. Molecular dynamics simulation showed that while binding of calmodulin to the supposed low-affinity binding site on CaMKII is compatible with closing (and hence, inactivation) of the kinase, and could even favour it, binding to the high-affinity site is not. Stochastic simulations of a biochemical model showed that the existence of two such binding sites, one of them accessible only in the active, open conformation, would be sufficient to explain calmodulin trapping by CaMKII. We can explain the effect of CaMKII autophosphorylation at Thr286 on calmodulin trapping: It stabilises the active state and therefore makes the high-affinity binding site accessible. Crucially, a model with only one binding site where calmodulin binding and CaMKII inactivation are strictly mutually exclusive cannot reproduce calmodulin trapping. One of the predictions of our study is that calmodulin binding in itself is not sufficient for CaMKII activation, although high-affinity binding of calmodulin is

Elucidating the mechanisms of cooperative calcium-calmodulin interactions: a structural systems biology approach

BACKGROUND: Calmodulin is an important multifunctional molecule that regulates the activities of a large number of proteins in the cell. Calcium binding induces conformational transitions in calmodulin that make it specifically active to particular target proteins. The precise mechanisms underlying calcium binding to calmodulin are still, however, quite poorly understood. RESULTS: In this study, we adopt a structural systems biology approach and develop a mathematical model to investigate various types of cooperative calcium-calmodulin interactions. We compare the predictions of our analysis with physiological dose-response curves taken from the literature, in order to provide a quantitative comparison of the effects of different mechanisms of cooperativity on calcium-calmodulin interactions. The results of our analysis reduce the gap between current understanding of intracellular calmodulin function at the structural level and physiological calcium-dependent calmodulin target activation experiments. CONCLUSION: Our model predicts that the specificity and selectivity of CaM target regulation is likely to be due to the following factors: variations in the target-specific Ca2+ dissociation and cooperatively effected dissociation constants, and variations in the number of Ca2+ ions required to bind CaM for target activation

Modulation of calmodulin lobes by different targets: an allosteric model with hemiconcerted conformational transitions

Calmodulin, the ubiquitous calcium-activated second messenger in eukaryotes, is an extremely versatile molecule involved in many biological processes: muscular contraction, synaptic plasticity, circadian rhythm, and cell cycle, among others. The protein is structurally organised into two globular lobes, joined by a flexible linker. Calcium modulates calmodulin activity by favoring a conformational transition of each lobe from a closed conformation to an open conformation. Most targets have a strong preference for one conformation over the other, and depending on the free calcium concentration in a cell, particular sets of targets will preferentially interact with calmodulin. In turn, targets can increase or decrease the calcium affinity of the calmodulin molecules to which they bind. Interestingly, experiments with the tryptic fragments showed that most targets have a much lower affinity for the N-lobe than for the C-lobe. Hence, the latter predominates in the formation of most calmodulin-target complexes. We showed that a relatively simple allosteric mechanism, based the classic MWC model, can capture the observed modulation of both the isolated C-lobe, and intact calmodulin, by individual targets. Moreover, our model can be naturally extended to study how the calcium affinity of a single pool of calmodulin is modulated by a mixture of competing targets in vivo

MODULATION OF THE CGMP-GATED CHANNEL BY CALCIUM

Calcium acting through calmodulin has been shown to regulate the affinity of cyclic nucleotide-gated channels expressed in cell lines. But is calmodulin the Ca-sensor that normally regulates these channels

Activation of type II calcium/calmodulin-dependent protein kinase by Ca^(2+)/calmodulin is inhibited by autophosphorylation of threonine within the calmodulin-binding domain

It is now well established that autophosphorylation of a threonine residue located next to each calmodulin-binding domain in the subunits of type II Ca^(2+)/calmodulin-dependent protein kinase causes the kinase to remain active, although at a reduced rate, after Ca^(2+) is removed from the reaction. This autophosphorylated form of the kinase is still sensitive to Ca2+/calmodulin, which is required for a maximum catalytic rate. After removal of Ca^(2+), new sites are autophosphorylated by the partially active kinase. Autophosphorylation of these sites abolishes sensitivity of the kinase to Ca^(2+)/calmodulin (Hashimoto, Y., Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1987) J. Biol. Chem. 262, 8051-8055). We have identified two pairs of homologous residues, Thr^(305) and Ser^(314) in the alpha subunit and Thr^(306) and Ser^(315) in the beta subunit, that are autophosphorylated only after removal of Ca^(2+) from an autophosphorylation reaction. The sites were identified by direct sequencing of labeled tryptic phosphopeptides isolated by reverse-phase high pressure liquid chromatography. Thr^(305-306) is rapidly dephosphorylated by purified protein phosphatases 1 and 2A, whereas Ser^(314-315) is resistant to dephosphorylation. We have shown by selective dephosphorylation that the presence of phosphate on Thr^(305-306) blocks sensitivity of the kinase to Ca^(2+)/calmodulin. In contrast, the presence of phosphate on Ser^(314-315) is associated with an increase in the Kact for Ca^(2+)/calmodulin of only about 2-fold, producing a relatively small decrease in sensitivity to Ca^(2+)/calmodulin

Modulation of the asymmetry of sea urchin sperm flagellar bending by calmodulin

Sea urchin spermatozoa demembranated with Triton X-100 in the presence of EGTA, termed potentially asymmetric, generate asymmetric bending waves in reactivation solutions containing EGTA. After they are converted to the potentially symmetric condition by extraction with Triton and millimolar Ca++, they generate symmetric bending waves in reactivation solutions containing EGTA. In the presence of EGTA, their asymmetry can be restored by addition of brain calmodulin or the concentrated supernatant obtained from extraction with Triton and millimolar Ca++. These extracts contain calmodulin, as assayed by gel electrophoresis, radioimmunoassay, activation of brain phosphodiesterase, and Ca++-dependent binding of asymmetry-restoring activity to a trifluorophenothiazine-affinity resin. Conversion to the potentially symmetric condition can also be achieved with trifluoperazine substituted for Triton during the exposure to millimolar Ca++, which suggests that the calmodulin-binding activity of Triton is important for this conversion. These observations suggest that the conversion to the potentially symmetric condition is the result of removal of some of the axonemal calmodulin and provide additional evidence for axonemal calmodulin as a mediator of the effect of Ca++ on the asymmetry of flagellar bending

Mechanical Stretching of Proteins: Calmodulin and Titin

Mechanical unfolding of several domains of calmodulin and titin is studied using a Go-like model with a realistic contact map and Lennard-Jones contact interactions. It is shown that this simple model captures the experimentally observed difference between the two proteins: titin is a spring that is tough and strong whereas calmodulin acts like a weak spring with featureless force-displacement curves. The difference is related to the dominance of the alpha secondary structures in the native structure of calmodulin. The tandem arrangements of calmodulin unwind simultaneously in each domain whereas the domains in titin unravel in a serial fashion. The sequences of contact events during unraveling are correlated with the contact order, i.e. with the separation between contact making amino acids along the backbone in the native state. Temperature is found to affect stretching in a profound way.Comment: To be published in a special bio-issue of Physica A; 14 figure

Conformational changes of calmodulin upon Ca2+ binding studied with a microfluidic mixer

A microfluidic mixer is applied to study the kinetics of calmodulin conformational changes upon Ca2+ binding. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive mixing and accesses time scales of tens of microseconds. The mixer is used in conjunction with multiphoton microscopy to examine the fast Ca2+-induced transitions of acrylodan-labeled calmodulin. We find that the kinetic rates of the conformational changes in two homologous globular domains differ by more than an order of magnitude. The characteristic time constants are ≈490 μs for the transitions in the C-terminal domain and ≈20 ms for those in the N-terminal domain of the protein. We discuss possible mechanisms for the two distinct events and the biological role of the stable intermediate, half-saturated calmodulin

Flagellar Radial Spoke Protein 2 Is a Calmodulin Binding Protein Required for Motility in \u3cem\u3eChlamydomonas reinhardtii\u3c/em\u3e

Genetic and morphological studies have revealed that the radial spokes regulate ciliary and flagellar bending. Functional and biochemical analysis and the discovery of calmodulin in the radial spokes suggest that the regulatory mechanism involves control of axonemal protein phosphorylation and calcium binding to spoke proteins. To identify potential regulatory proteins in the radial spoke, in-gel kinase assays were performed on isolated axonemes and radial spoke fractions. The results indicated that radial spoke protein 2 (RSP2) can bind ATP and transfer phosphate in vitro. RSP2 was cloned and mapped to the PF24 locus, a gene required for motility. Sequencing revealed that pf24 contains a point mutation converting the first ATG to ATA, resulting in only trace amounts of RSP2 and confirming the RSP2 mapping. Surprisingly, the sequence does not include signature domains for conventional kinases, indicating that RSP2 may not perform as a protein kinase in vivo. However, the predicted RSP2 protein sequence contains Ca2+-dependent calmodulin binding motifs and a GAF domain, a domain found in diverse signaling proteins for binding small ligands including cyclic nucleotides. As predicted from the sequence, recombinant RSP2 binds calmodulin in a calcium-dependent manner. We postulate that RSP2 is a regulatory subunit of the radial spoke involved in localization of calmodulin for control of motility

Calmodulin in Complex with Proteins and Small Molecule Ligands: Operating with the Element of Surprise. Implications for Structure-Based Drug Design

Calmodulin plays a role in several life processes, its flexibility allows binding of a number of different ligands from small molecules to amphiphilic peptide helices and proteins. Through the diversity of its functions, it is quite difficult to find new drugs, which bind to calmodulin as a target. We present available structural information on the protein, obtained by X-ray diffraction, nuclear magnetic resonance spectroscopy and molecular modeling and try to derive some conclusions on structureactivity relationships