Real-time single-molecule imaging of CaMKII-calmodulin interactions.

The binding of calcium/calmodulin (CAM) to calcium/calmodulin-dependent protein kinase II (CaMKII) initiates an ATP-driven cascade that triggers CaMKII autophosphorylation. The autophosphorylation in turn increases the CaMKII affinity for CAM. Here, we studied the ATP dependence of CAM association with the actin-binding CaMKIIß isoform using single-molecule total internal reflection fluorescence microscopy. Rhodamine-CAM associations/dissociations to surface-immobilized Venus-CaMKIIß were resolved with 0.5 s resolution from video records, batch-processed with a custom algorithm. CAM occupancy was determined simultaneously with spot-photobleaching measurement of CaMKII holoenzyme stoichiometry. We show the ATP-dependent increase of the CAM association requires dimer formation for both the α and ß isoforms. The study of mutant ß holoenzymes revealed that the ATP-dependent increase in CAM affinity results in two distinct states. The phosphorylation-defective (T287.306-307A) holoenzyme resides only in the low-affinity state. CAM association is further reduced in the T287A holoenzyme relative to T287.306-307A. In the absence of ATP, the affinity of CAM for the T287.306-307A mutant and the wild-type monomer are comparable. The affinity of the ATP-binding impaired (K43R) mutant is even weaker. In ATP, the K43R holoenzyme resides in the low-affinity state. The phosphomimetic mutant (T287D) resides only in a 1000-fold higher-affinity state, with mean CAM occupancy of more than half of the 14-mer holoenzyme stoichiometry in picomolar CAM. ATP promotes T287D holoenzyme disassembly but does not elevate CAM occupancy. Single Poisson distributions characterized the ATP-dependent CAM occupancy of mutant holoenzymes. In contrast, the CAM occupancy of the wild-type population had a two-state distribution with both low- and high-affinity states represented. The low-affinity state was the dominant state, a result different from published in vitro assays. Differences in assay conditions can alter the balance between activating and inhibitory autophosphorylation. Bound ATP could be sufficient for CaMKII structural function, while antagonistic autophosphorylations may tune CaMKII kinase-regulated action-potential frequency decoding in vivo.

Controlling the interaction between CaMKII and Calmodulin with a photocrosslinking unnatural amino acid.

Using unnatural amino acid mutagenesis, we made a mutant of CaMKII that forms a covalent linkage to Calmodulin upon illumination by UV light. Like wild-type CaMKII, the L308BzF mutant stoichiometrically binds to Calmodulin, in a calcium-dependent manner. Using this construct, we demonstrate that Calmodulin binding to CaMKII, even under these stochiometric conditions, does not perturb the CaMKII oligomeric state. Furthermore, we were able to achieve activation of CaMKII L308BzF by UV-induced binding of Calmodulin, which, once established, is further insensitive to calcium depletion. In addition to the canonical auto-inhibitory role of the regulatory segment, inter-subunit crosslinking in the absence of CaM indicates that kinase domains and regulatory segments are substantially mobile in basal conditions. Characterization of CaMKIIL308BzF in vitro, and its expression in mammalian cells, suggests it could be a promising candidate for control of CaMKII activity in mammalian cells with light.

LTP induction by structural rather than enzymatic functions of CaMKII.

Learning and memory are thought to require hippocampal long-term potentiation (LTP), and one of the few central dogmas of molecular neuroscience that has stood undisputed for more than three decades is that LTP induction requires enzymatic activity of the Ca2+/calmodulin-dependent protein kinase II (CaMKII)1-3. However, as we delineate here, the experimental evidence is surprisingly far from conclusive. All previous interventions inhibiting enzymatic CaMKII activity and LTP4-8 also interfere with structural CaMKII roles, in particular binding to the NMDA-type glutamate receptor subunit GluN2B9-14. Thus, we here characterized and utilized complementary sets of new opto-/pharmaco-genetic tools to distinguish between enzymatic and structural CaMKII functions. Several independent lines of evidence demonstrated LTP induction by a structural function of CaMKII rather than by its enzymatic activity. The sole contribution of kinase activity was autoregulation of this structural role via T286 autophosphorylation, which explains why this distinction has been elusive for decades. Directly initiating the structural function in a manner that circumvented this T286 role was sufficient to elicit robust LTP, even when enzymatic CaMKII activity was blocked.

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. METHODS: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. RESULTS: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. CONCLUSIONS: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

Conserved and divergent features of neuronal CaMKII holoenzyme structure, function, and high-order assembly.

Neuronal CaMKII holoenzymes (α and ß isoforms) enable molecular signal computation underlying learning and memory but also mediate excitotoxic neuronal death. Here, we provide a comparative analysis of these signaling devices, using single-particle electron microscopy (EM) in combination with biochemical and live-cell imaging studies. In the basal state, both isoforms assemble mainly as 12-mers (but also 14-mers and even 16-mers for the ß isoform). CaMKIIα and ß isoforms adopt an ensemble of extended activatable states (with average radius of 12.6 versus 16.8 nm, respectively), characterized by multiple transient intra- and inter-holoenzyme interactions associated with distinct functional properties. The extended state of CaMKIIß allows direct resolution of intra-holoenzyme kinase domain dimers. These dimers could enable cooperative activation by calmodulin, which is observed for both isoforms. High-order CaMKII clustering mediated by inter-holoenzyme kinase domain dimerization is reduced for the ß isoform for both basal and excitotoxicity-induced clusters, both in vitro and in neurons.

CaMKIIα-driven, phosphatase-checked postsynaptic plasticity via phase separation.

Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) is essential for synaptic plasticity and learning by decoding synaptic Ca2+ oscillations. Despite decades of extensive research, new mechanisms underlying CaMKIIα's function in synapses are still being discovered. Here, we discover that Shank3 is a specific binding partner for autoinhibited CaMKIIα. We demonstrate that Shank3 and GluN2B, via combined actions of Ca2+ and phosphatases, reciprocally bind to CaMKIIα. Under basal condition, CaMKIIα is recruited to the Shank3 subcompartment of postsynaptic density (PSD) via phase separation. Rise of Ca2+ concentration induces GluN2B-mediated recruitment of active CaMKIIα and formation of the CaMKIIα/GluN2B/PSD-95 condensates, which are autonomously dispersed upon Ca2+ removal. Protein phosphatases control the Ca2+-dependent shuttling of CaMKIIα between the two PSD subcompartments and PSD condensate formation. Activation of CaMKIIα further enlarges the PSD assembly and induces structural LTP. Thus, Ca2+-induced and phosphatase-checked shuttling of CaMKIIα between distinct PSD nano-domains can regulate phase separation-mediated PSD assembly and synaptic plasticity.

CaMKIIß in Neuronal Development and Plasticity: An Emerging Candidate in Brain Diseases.

The calcium/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous and central player in Ca2+ signaling that is best known for its functions in the brain. In particular, the α isoform of CaMKII has been the subject of intense research and it has been established as a central regulator of neuronal plasticity. In contrast, little attention has been paid to CaMKIIß, the other predominant brain isoform that interacts directly with the actin cytoskeleton, and the functions of CaMKIIß in this organ remain largely unexplored. However, recently, the perturbation of CaMKIIß expression has been associated with multiple neuropsychiatric and neurodevelopmental diseases, highlighting CAMK2B as a gene of interest. Herein, after highlighting the main structural and expression differences between the α and ß isoforms, we will review the specific functions of CaMKIIß, as described so far, in neuronal development and plasticity, as well as its potential implication in brain diseases.

Coordination between Calcium/Calmodulin-Dependent Protein Kinase II and Neuronal Nitric Oxide Synthase in Neurons.

Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is highly abundant in the brain and exhibits broad substrate specificity, thereby it is thought to participate in the regulation of neuronal death and survival. Nitric oxide (NO), produced by neuronal NO synthase (nNOS), is an important neurotransmitter and plays a role in neuronal activity including learning and memory processes. However, high levels of NO can contribute to excitotoxicity following a stroke and neurodegenerative disease. Aside from NO, nNOS also generates superoxide which is involved in both cell injury and signaling. CaMKII is known to activate and translocate from the cytoplasm to the post-synaptic density in response to neuronal activation where nNOS is predominantly located. Phosphorylation of nNOS at Ser847 by CaMKII decreases NO generation and increases superoxide generation. Conversely, NO-induced S-nitrosylation of CaMKII at Cys6 is a prominent determinant of the CaMKII inhibition in ATP competitive fashion. Thus, the "cross-talk" between CaMKII and NO/superoxide may represent important signal transduction pathways in brain. In this review, we introduce the molecular mechanism of and pathophysiological role of mutual regulation between CaMKII and nNOS in neurons.

Supramolecular structure, in vivo biological activities and molecular-docking-based potential cardiotoxic exploration of aconine hydrochloride monohydrate as a novel salt form.

Despite the high profile of aconine in WuTou injection, there has been no preparative technology or structural studies of its salt as the pharmaceutical product. The lack of any halide salt forms is surprising as aconine contains a tertiary nitrogen atom. In this work, aconine was prepared from the degradation of aconitine in Aconiti kusnezoffii radix (CaoWu). A green chemistry technique was applied to enrich the lipophilic-poor aconine. Reaction of aconine with hydrochloride acid resulted in protonation of the nitrogen atom and gave a novel salt form (C25H42NO9+·Cl-·H2O; aconine hydrochloride monohydrate, AHM), whose cation in the crystal structure was elucidated based on extensive spectroscopic and X-ray crystallographic analyses. The AHM crystal had a Z' = 3 structure with three independent cation-anion pairs, with profound conformational differences among the aconine cations. The central framework of each aconine cation was compared with that of previously reported aconitine, proving that protonation of the nitrogen atom induced the structure rearrangement. In the crystal of AHM, aconine cations, chloride anions and water molecules interacted through inter-species O-H...Cl and O-H...O hydrogen bonds; this complex hydrogen-bonding network stabilizes the supramolecular structure. The seriously disordered solvent molecules were treated using the PLATON SQUEEZE procedure [Spek (2015). Acta Cryst. C71, 9-18] and their atoms were therefore omitted from the refinement. Bioactivity studies indicated that AHM promoted in vitro proliferative activities of RAW264.7 cells. Molecular docking suggested AHM could target cardiotoxic protein through the hydrogen-bonding interactions. The structural confirmation of AHM offers a rational approach for improving the pharmaceutical technology of WuTou injection.

The CaMKII K42M and K42R mutations are equivalent in suppressing kinase activity and targeting.

CaMKII is an important mediator of forms of synaptic plasticity that are thought to underly learning and memory. The CaMKII mutants K42M and K42R have been used interchangeably as research tools, although some reported phenotypic differences suggest that they may differ in the extent to which they impair ATP binding. Here, we directly compared the two mutations at the high ATP concentrations that exist within cells (~4 mM). We found that both mutations equally blocked GluA1 phosphorylation in vitro and GluN2B binding within cells. Both mutations also reduced but did not completely abolish CaMKII T286 autophosphorylation in vitro or CaMKII movement to excitatory synapses in neurons. Thus, despite previously suggested differences, both mutations appear to interfere with ATP binding to the same extent.

The CaMKII phosphorylation site Thr1604 in the CaV1.2 channel is involved in pathological myocardial hypertrophy in rats.

Residue Thr1604 in the CaV1.2 channel is a Ca2+/calmodulin dependent protein kinase II (CaMKII) phosphorylation site, and its phosphorylation status maintains the basic activity of the channel. However, the role of CaV1.2 phosphorylation at Thr1604 in myocardial hypertrophy is incompletely understood. Isoproterenol (ISO) was used to induce cardiomyocyte hypertrophy, and autocamtide-2-related inhibitory peptide (AIP) was added as a treatment. Rats in a myocardial hypertrophy development model were subcutaneously injected with ISO for two or three weeks. The heart and left ventricle weights, each of which were normalized to the body weight and cross-sectional area of the myocardial cells, were used to describe the degree of hypertrophy. Protein expression levels were detected by western blotting. CaMKII-induced CaV1.2 (Thr1604) phosphorylation (p-CaV1.2) was assayed by coimmunoprecipitation. The results showed that CaMKII, HDAC, MEF2 C, and atrial natriuretic peptide (ANP) expression was increased in the ISO group and downregulated by AIP treatment in vitro. There was no difference in the expression of these proteins between the ISO 2-week group and the ISO 3-week group in vivo. CaV1.2 channel expression did not change, but p-CaV1.2 expression was increased after ISO stimulation and decreased by AIP. In the rat model, p-CaV1.2 levels and CaMKII activity were much higher in the ISO 3-week group than in the ISO 2-week group. CaMKII-induced CaV1.2 channel phosphorylation at residue Thr1604 may be one of the key features of myocardial hypertrophy and disease development.Abbreviations: CaMKII: Ca2+/calmodulin dependent protein kinase II; p-CaMKII: autophosphorylated Ca2+/calmodulin dependent protein kinase II; CaM: calmodulin; AIP: autocamtide-2-related inhibitory peptide; ECC: excitation-contraction coupling; ISO: isoproterenol; BW: body weight; HW: heart weight; LVW: left ventricle weight; HDAC: histone deacetylase; p-HDAC: phosphorylated histone deacetylase; MEF2C: myocyte-specific enhancer factor 2C; ANP: atrial natriuretic peptide; PKC: protein kinase C.

Characterization of CaMKIIα holoenzyme stability.

Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation and other Ca2+ -dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKIIα using a combination of differential scanning calorimetry (DSC), X-ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent Tm = 36°C), which is slightly stabilized by ATP/MgCl2 binding (apparent Tm = 40°C) and significantly stabilized by regulatory segment binding (apparent Tm = 60°C). We crystallized the kinase domain of CaMKII bound to p-coumaric acid in the active site. This structure reveals solvent-exposed hydrophobic residues in the substrate-binding pocket, which are normally buried in the autoinhibited structure when the regulatory segment is present. This likely accounts for the large stabilization that we observe in DSC measurements comparing the kinase alone with the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent Tm ~ 90°C), and the holoenzyme structure has multiple unfolding transitions ranging from ~60°C to 100°C. Using MP, we compared a CaMKIIα holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared with the hub domain alone. We conclude that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. These data support a model where domains within the holoenzyme interact.

Stitching the synapse: Cross-linking mass spectrometry into resolving synaptic protein interactions.

Synaptic transmission is the predominant form of communication in the brain. It requires functionally specialized molecular machineries constituted by thousands of interacting synaptic proteins. Here, we made use of recent advances in cross-linking mass spectrometry (XL-MS) in combination with biochemical and computational approaches to reveal the architecture and assembly of synaptic protein complexes from mouse brain hippocampus and cerebellum. We obtained 11,999 unique lysine-lysine cross-links, comprising connections within and between 2362 proteins. This extensive collection was the basis to identify novel protein partners, to model protein conformational dynamics, and to delineate within and between protein interactions of main synaptic constituents, such as Camk2, the AMPA-type glutamate receptor, and associated proteins. Using XL-MS, we generated a protein interaction resource that we made easily accessible via a web-based platform (http://xlink.cncr.nl) to provide new entries into exploration of all protein interactions identified.

Glu60 of α-Calcium/calmodulin dependent protein kinase II mediates crosstalk between the regulatory T-site and protein substrate binding region of the active site.

Memory formation transpires to be by activation and persistent modification of synapses. A chain of biochemical events accompany synaptic activation and culminate in memory formation. These biochemical events are steered by interplay and modulation of various synaptic proteins, achieved by conformational changes and phosphorylation/dephosphorylation of these proteins. Calcium/calmodulin dependent protein kinase II (CaMKII) and N-methyl-d-aspartate receptors (NMDARs) are synaptic proteins whose interactions play a pivotal role in learning and memory process. Catalytic activity of CaMKII is modulated upon its interaction with the GluN2B subunit of NMDAR. The structural basis of this interaction is not clearly understood. We have investigated the role of Glu60 of α-CaMKII, a conserved residue present in the ATP binding region of kinases, in the regulation of catalysis of CaMKII by GluN2B. Mutation of Glu60 to Gly exerts different effects on the kinetic parameters of phosphorylation of GluN2B and GluN2A, of which only GluN2B binds to the T-site of CaMKII. GluN2B induced modulation of the kinetic parameters of peptide substrate was altered in the E60G mutant. The mutation almost abolished the modulation of the apparent Km value for protein substrate. However, although kinetic parameters for ATP were altered by mutating Glu60, modulation of the apparent Km value for ATP by GluN2B seen in WT was exhibited by the E60G mutant of α-CaMKII. Hence our results posit that the communication of the T-site of CaMKII with protein substrate binding region of active site is mediated through Glu60 while the communication of the T-site with the ATP binding region is not entirely dependent on Glu60.

Flexible linkers in CaMKII control the balance between activating and inhibitory autophosphorylation.

The many variants of human Ca2+/calmodulin-dependent protein kinase II (CaMKII) differ in the lengths and sequences of disordered linkers connecting the kinase domains to the oligomeric hubs of the holoenzyme. CaMKII activity depends on the balance between activating and inhibitory autophosphorylation (on Thr 286 and Thr 305/306, respectively, in the human α isoform). Variation in the linkers could alter transphosphorylation rates within a holoenzyme and the balance of autophosphorylation outcomes. We show, using mammalian cell expression and a single-molecule assay, that the balance of autophosphorylation is flipped between CaMKII variants with longer and shorter linkers. For the principal isoforms in the brain, CaMKII-α, with a ~30 residue linker, readily acquires activating autophosphorylation, while CaMKII-ß, with a ~200 residue linker, is biased towards inhibitory autophosphorylation. Our results show how the responsiveness of CaMKII holoenzymes to calcium signals can be tuned by varying the relative levels of isoforms with long and short linkers.

The cardiac CaMKII-Nav1.5 relationship: From physiology to pathology.

The SCN5A gene encodes Nav1.5, which, as the cardiac voltage-gated Na+ channel's pore-forming α subunit, is crucial for the initiation and propagation of atrial and ventricular action potentials. The arrhythmogenic propensity of inherited SCN5A mutations implicates the Na+ channel in determining cardiomyocyte excitability under normal conditions. Cytosolic kinases have long been known to alter the kinetic profile of Nav1.5 inactivation via phosphorylation of specific residues. Recent substantiation of both the role of calmodulin-dependent kinase II (CaMKII) in modulating the properties of the Nav1.5 inactivation gate and the significant rise in oxidation-dependent autonomous CaMKII activity in structural heart disease has raised the possibility of a novel pathway for acquired arrhythmias - the CaMKII-Nav1.5 relationship. The aim of this review is to: (1) outline the relationship's translation from physiological adaptation to pathological vicious circle; and (2) discuss the relative merits of each of its components as pharmacological targets.

Cellular Adaptation Relies on Regulatory Proteins Having Episodic Memory: Proteins Modulate Cell Metabolism and Reproduction by Remembering, Transmitting, and Using Data on the Environment.

The ability to memorize changes in the environment is present at all biological levels, from social groups and individuals, down to single cells. Trans-generational memory is embedded subcellularly through genetic and epigenetic mechanisms. Evidence that cells process and remember features of the immediate environment using protein sensors is reviewed. It is argued that this mnemonic ability is encapsulated within the protein conformational space and lasts throughout its lifetime, which can overlap with the lifespan of the organism. Means to determine diachronic changes in protein activity are presented.

A multi-state model of the CaMKII dodecamer suggests a role for calmodulin in maintenance of autophosphorylation.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) accounts for up to 2 percent of all brain protein and is essential to memory function. CaMKII activity is known to regulate dynamic shifts in the size and signaling strength of neuronal connections, a process known as synaptic plasticity. Increasingly, computational models are used to explore synaptic plasticity and the mechanisms regulating CaMKII activity. Conventional modeling approaches may exclude biophysical detail due to the impractical number of state combinations that arise when explicitly monitoring the conformational changes, ligand binding, and phosphorylation events that occur on each of the CaMKII holoenzyme's subunits. To manage the combinatorial explosion without necessitating bias or loss in biological accuracy, we use a specialized syntax in the software MCell to create a rule-based model of a twelve-subunit CaMKII holoenzyme. Here we validate the rule-based model against previous experimental measures of CaMKII activity and investigate molecular mechanisms of CaMKII regulation. Specifically, we explore how Ca2+/CaM-binding may both stabilize CaMKII subunit activation and regulate maintenance of CaMKII autophosphorylation. Noting that Ca2+/CaM and protein phosphatases bind CaMKII at nearby or overlapping sites, we compare model scenarios in which Ca2+/CaM and protein phosphatase do or do not structurally exclude each other's binding to CaMKII. Our results suggest a functional mechanism for the so-called "CaM trapping" phenomenon, wherein Ca2+/CaM may structurally exclude phosphatase binding and thereby prolong CaMKII autophosphorylation. We conclude that structural protection of autophosphorylated CaMKII by Ca2+/CaM may be an important mechanism for regulation of synaptic plasticity.

CaM Kinase: Still Inspiring at 40.

The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) was touted as a memory molecule, even before its involvement in long-term potentiation (LTP) was shown. The enzyme has not disappointed, with subsequent demonstrations of remarkable structural and regulatory properties. Its neuronal functions now extend to long-term depression (LTD), and last year saw the first direct evidence for memory storage by CaMKII. Although CaMKII may have taken the spotlight, it is a member of a large family of diverse and interesting CaM kinases. Our aim is to place CaMKII in context of the other CaM kinases and then review certain aspects of this kinase that are of current interest.