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The small IQ motif proteins PEP-19 (62 amino acids) and RC3 (78 amino acids) greatly accelerate the rates of Ca2+ binding to sites III and IV in the C-domain of calmodulin (CaM). We show here that PEP-19 decreases the degree of cooperativity of Ca2+ binding to sites III and IV and we present a model showing that this could increase Ca2+ binding rate constants. Comparative sequence analysis showed that residues 28 to 58 from PEP-19 are conserved in other proteins. This region includes the IQ motif (amino acids 39-62), and an adjacent acidic cluster of amino acids (amino acids 28-40). A synthetic peptide spanning residues 28-62 faithfully mimics intact PEP-19 with respect to increasing the rates of Ca2+ association and dissociation, as well as binding preferentially to the C-domain of CaM. In contrast, a peptide encoding only the core IQ motif does not modulate Ca2+ binding, and binds to multiple sites on CaM. A peptide that includes only the acidic region does not bind to CaM. These results show that PEP-19 has a novel acidic/IQ CaM regulatory motif in which the IQ sequence provides a targeting function that allows binding of PEP-19 to CaM, whereas the acidic residues modify the nature of this interaction, and are essential for modulating Ca2+ binding to the C-domain of CaM. [ABSTRACT FROM AUTHOR]

The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca2+ binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca2+-calmodulin should increase the affinity of calmodulin for Ca2+, while proteins that bind to apo-calmodulin should decrease its affinity for Ca2+. We quantified the effects on Ca2+-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca2+- and apo-calmodulin, and aaCaM kinase II, which binds selectively to Ca2+-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca2+, whereas CaM kinase II increases the calmodulin affinity for Ca2+. Specifically, RC3 increases the rate of Ca2+ dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca2+ association. Conversely, CaM kinase II decreases the rates of dissociation of Ca2+ from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr286 induces a further decrease in the rates of Ca2+ dissociation. RC3 dampens the effects of CaM kinase II on Ca2+ dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca2+-calmodulin target complex. This work indicates that the Ca2+ binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca2+-calmodulin target complexes at the end of a Ca2+ signal. [ABSTRACT FROM AUTHOR]

Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2) constitute a family of similar calcium sensors that regulate synaptic plasticity. They are both guanine exchange factors that contain a very similar set of functional domains, including N-terminal pleckstrin homology, coiled-coil, and calmodulin-binding IQ domains and C-terminal Dbl homology Rac-activating domains, Ras-exchange motifs, and CDC25 Ras-activating domains. Nevertheless, they regulate different forms of synaptic plasticity. Although both GRF proteins transduce calcium signals emanating from NMDA-type glutamate receptors in the CA1 region of the hippocampus, GRF1 promotes LTD, whereas GRF2 promotes ϑ-burst stimulation-induced LTP (TBS-LTP). GRF1 can also mediate high frequency stimulation-induced LTP (HFS-LTP) in mice over 2-months of age, which involves calcium-permeable AMPA-type glutamate receptors. To add to our understanding of how proteins with similar domains can have different functions, WT and various chimeras between GRF1 and GRF2 proteins were tested for their abilities to reconstitute defective LTP and/or LTD in the CA1 hippocampus of Grf1/Grf2 double knock-out mice. These studies revealed a critical role for the GRF2 CDC25 domain in the induction of TBS-LTP by GRF proteins. In contrast, the N-terminal pleckstrin homology and/or coiled-coil domains of GRF1 are key to the induction of HFSLTP by GRF proteins. Finally, the IQ motif of GRF1 determines whether a GRF protein can induce LTD. Overall, these findings show that for the three forms of synaptic plasticity that are regulated by GRF proteins in the CA1 hippocampus, specificity is encoded in only one or two domains, and a different set of domains for each form of synaptic plasticity. [ABSTRACT FROM AUTHOR]

Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca2+ but to a lesser extent (2-3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a >10-fold decrease in the affinity of Ca2+ binding to the C-terminal domain of CaM with an associated increase in the Ca2+ dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca2+ binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca2+-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca2+ binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca2+-bound CaM and that although Ca2+ binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca2+ binding to the C-terminal lobe of CaM. [ABSTRACT FROM AUTHOR]

Ca2+-Calmodulin-dependent protein kinase II (CaMKI) is an abundant synaptic protein that was recently shown to regulate the organization of actin filaments leading to structural modifications of synapses. CaMKII is a dodecameric complex with a special architecture that provides it with unique potential for organizing the actin cytoskeleton. We report using biochemical assays that the β isoform of CaMKII binds to and bundles actin filaments, and the disposition of βCaMKII within the actin bundles was revealed by cryoelectron tomography. In addition, βCaMKII was found to inhibit actin polymerization, suggesting that it either serves as a capping protein or binds monomeric actin, reducing the amount of freely available monomers to nucleate polymer assembly. By means of fluorescent cross-correlation spectroscopy, we determined that βCaMKII does indeed bind to monomeric actin, reaching saturation at a stoichiometry of 12:1 actin monomers per βCaMKII holoenzyme with a binding constant of 2.4 x 105 M-1. In cells, βCaMKII has a dual functional role; it can sequester monomeric actin to reduce actin polymerization and can also bundle actin filaments. Together, these effects would impact both the dynamics of actin filament assembly and enhance the rigidity of the filaments once formed, significantly impacting the structure of synapses. [ABSTRACT FROM AUTHOR]

Summary: The structure of the endosomal-associated protein, Hrs, has been determined with cryo-electron microscopy. Hrs interacts with a number of proteins, including SNAP-25 and STAM1, forming a complex that binds ubiquitin moieties. Analytical ultracentrifugation studies revealed that Hrs exists as a hexamer. The symmetry and the structure of the hexameric form of Hrs were determined with the single-particle reconstruction method. Hrs comprises three antiparallel dimers with a central core and distinct caps on either end. Crystal structures of VHS and FYVE domains fit into the Hrs end caps in the EM density map. Thus, the location of domains that interact with the endosomal membrane, the VHS, FYVE, and C-terminal domains, facilitates the anchorage of Hrs to the membrane, initiating the functional processes of Hrs on the endosome. Based on our model, the Hrs hexamer interacts with the membrane and acts as a “master molecule” that presents multiple sites for protein binding. [Copyright &y& Elsevier]

Ca2+-calmodulin-dependent protein kinase II (CAMkinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (α, β, γ, and δ) in mammalian cells. We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products. Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21-25 Å revealed that all four isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core. At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes. Enzymatic analyses showed that the isoforms were similar in their Km for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca2+-calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation. Interestingly, the rank order of CaM binding affinity (γ > β > δ > α) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (β > γ > δ > α). Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform. [ABSTRACT FROM AUTHOR]

IQ motifs are found in diverse families of calmodulin (CaM)-binding proteins. Some of these, like PEP-19 and RC3, are highly abundant in neuronal tissues, but being devoid of catalytic activity, their biological roles are not understood. We hypothesized that these IQ motif proteins might have unique effects on the Ca[sup 2+] binding properties of CaM, since they bind to CaM in the presence or absence of Ca[sup 2+]. Here we show that PEP-19 accelerates by 40 to 50-fold both the slow association and dissociation of Ca[sup 2+] from the C-domain of free CaM, and we identify the sites of interaction between CaM and PEP-19 using NMR. Importantly, we demonstrate that PEP-19 can also increase the rate of dissociation of Ca[sup 2+] from CaM when bound to intact CaM-dependent protein kinase II. Thus, PEP-19, and presumably similar members of the IQ family of proteins, has the potential to alter the Ca[sup 2+]-binding dynamics of free CaM and CaM that is bound to other target proteins. Since Ca[sup 2+] binding to the C-domain of CaM is the rate-limiting step for activation of CaM-dependent enzymes, the data reveal a new concept of importance in understanding the temporal dynamics of Ca[sup 2+]-dependent cell signaling. [ABSTRACT FROM AUTHOR]