Novel Variant Causing Calmodulinopathy With Variable Expressivity in a 4-Generation Family.

Background: CaM (calmodulin), encoded by 3 separate genes (, , and ), is a multifunctional Ca-binding protein involved in many signal transduction events including ion channel regulation. CaM variants may present with early-onset long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia, or sudden cardiac death. Most reported variants occurred de novo.

Na1.2 EFL domain allosterically enhances Ca binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD.

Neuronal voltage-gated sodium channel Na1.2 C-terminal domain (CTD) binds calmodulin (CaM) constitutively at its IQ motif. A solution structure (6BUT) and other NMR evidence showed that the CaM N domain (CaM) is structurally independent of the C-domain (CaM) whether CaM is bound to the Na1.

Ca-saturated calmodulin binds tightly to the N-terminal domain of A-type fibroblast growth factor homologous factors.

Voltage-gated sodium channels (Nas) are tightly regulated by multiple conserved auxiliary proteins, including the four fibroblast growth factor homologous factors (FGFs), which bind the Na EF-hand like domain (EFL), and calmodulin (CaM), a multifunctional messenger protein that binds the Na IQ motif. The EFL domain and IQ motif are contiguous regions of Na cytosolic C-terminal domains (CTD), placing CaM and FGF in close proximity. However, whether the FGFs and CaM act independently, directly associate, or operate through allosteric interactions to regulate channel function is unknown.

MICAL1 constrains cardiac stress responses and protects against disease by oxidizing CaMKII.

Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII.

Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels Na1.1, Na1.4 and Na1.7.

Human voltage-gated sodium (Na) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane β-subunits. Na pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis.

The Nkd EF-hand domain modulates divergent wnt signaling outputs in zebrafish.

Wnt proteins regulate diverse biological responses by initiating two general outcomes: β-catenin-dependent transcription and β-catenin-independent activation of signaling cascades, the latter including modulation of calcium and regulation of cytoskeletal dynamics (Planar Cell Polarity, PCP). It has been difficult to elucidate the mechanisms by which Wnt signals are directed to effect one or the other outcome due to shared signaling proteins between the β-catenin-dependent and -independent pathways, such as the Dishevelled binding protein Naked. While all Naked paralogs contain a putative calcium-binding domain, the EF-Hand, Drosophila Naked does not bind calcium.

Backbone resonance assignments of complexes of human voltage-dependent sodium channel Na1.2 IQ motif peptide bound to apo calmodulin and to the C-domain fragment of apo calmodulin.

Human voltage-gated sodium channel Na1.2 has a single pore-forming α-subunit and two transmembrane β-subunits. Expressed primarily in the brain, Na1.

Backbone and side-chain resonance assignments of (Ca)-calmodulin bound to beta calcineurin A CaMBD peptide.

Calcineurin (CaN) is a heterodimeric and highly conserved serine/threonine phosphatase (PP2B) that plays a critical role in coupling calcium signals to physiological processes including embryonic cardiac development, NF-AT-regulated gene expression in immune responses, and apoptosis. The catalytic subunit (CaN) has three isoforms (α, β, and γ,) in humans and seven isoforms in Paramecium. In all eukaryotes, the EF-hand protein calmodulin (CaM) regulates CaN activity in a calcium-dependent manner.

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel Na1.2.

Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel Na1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM.

Opposing orientations of the anti-psychotic drug trifluoperazine selected by alternate conformations of M144 in calmodulin.

The anti-psychotic drug trifluoperazine (TFP) is an antagonist observed to bind to calcium-saturated calmodulin ((Ca(2+) )4 -CaM) at ratios of 1:1 (1CTR), 2:1 (1A29), and 4:1 (1LIN). Each structure contains one TFP bound in the hydrophobic cleft of the C-domain of CaM. However, the orientation of the trifluoromethyl (CF3 ) moiety differs among them: it is buried in the C-domain cleft of 1A29 and 1LIN, but protrudes from 1CTR.

Calcium-dependent energetics of calmodulin domain interactions with regulatory regions of the Ryanodine Receptor Type 1 (RyR1).

Calmodulin (CaM) allosterically regulates the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1): apo CaM activates the channel, while (Ca(2+))4-CaM inhibits it. CaM-binding RyR1 residues 1975-1999 and 3614-3643 were proposed to allow CaM to bridge adjacent RyR1 subunits. Fluorescence anisotropy titrations monitored the binding of CaM and its domains to peptides encompassing hRyR(11975-1999) or hRyR1(3614-3643).

Calmodulin and PI(3,4,5)P₃ cooperatively bind to the Itk pleckstrin homology domain to promote efficient calcium signaling and IL-17A production.

Precise regulation of the kinetics and magnitude of Ca(2+) signaling enables this signal to mediate diverse responses, such as cell migration, differentiation, vesicular trafficking, and cell death. We showed that the Ca(2+)-binding protein calmodulin (CaM) acted in a positive feedback loop to potentiate Ca(2+) signaling downstream of the Tec kinase family member Itk. Using NMR (nuclear magnetic resonance), we mapped CaM binding to two loops adjacent to the lipid-binding pocket within the Itk pleckstrin homology (PH) domain.

Introduction: twenty five years of the Gibbs Conference on Biothermodynamics.

In 2011, the Gibbs Conference on Biothermodynamics will celebrate its 25th anniversary. Since the inaugural meeting in 1987, it has brought together laboratories that lived, breathed and argued about the molecular logic of macromolecular machines. The participants have a deep commitment to understanding the nature of physico-chemical forces that govern regulation of biological systems, and share a passion for applying linkage theory.

Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of Ca(V)1.2.

Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca(2+) channel (Ca(V)1.2) regulates Ca(2+) entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with Ca(V)1.

Structural and energetic determinants of apo calmodulin binding to the IQ motif of the Na(V)1.2 voltage-dependent sodium channel.

The neuronal voltage-dependent sodium channel (Na(v)1.2), essential for generation and propagation of action potentials, is regulated by calmodulin (CaM) binding to the IQ motif in its α subunit. A peptide (Na(v)1.

Recognition of β-calcineurin by the domains of calmodulin: thermodynamic and structural evidence for distinct roles.

Calcineurin (CaN, PP2B, PPP3), a heterodimeric Ca(2+)-calmodulin-dependent Ser/Thr phosphatase, regulates swimming in Paramecia, stress responses in yeast, and T-cell activation and cardiac hypertrophy in humans. Calcium binding to CaN(B) (the regulatory subunit) triggers conformational change in CaN(A) (the catalytic subunit). Two isoforms of CaN(A) (α, β) are both abundant in brain and heart and activated by calcium-saturated calmodulin (CaM).

Allosteric effects of the antipsychotic drug trifluoperazine on the energetics of calcium binding by calmodulin.

Trifluoperazine (TFP; Stelazine) is an antagonist of calmodulin (CaM), an essential regulator of calcium-dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, and 1LIN) show TFP bound to (Ca(2+))(4)-CaM in ratios of 1, 2, or 4 TFP per CaM.

Thermodynamics and conformational change governing domain-domain interactions of calmodulin.

Calmodulin (CaM) is a small (148 amino acid), ubiquitously expressed eukaryotic protein essential for Ca(2+) regulation and signaling. This highly acidic polypeptide (pI<4) has two homologous domains (N and C), each consisting of two EF-hand Ca(2+)-binding sites. Despite significant homology, the domains have intrinsic differences in their Ca(2+)-binding properties and separable roles in regulating physiological targets such as kinases and ion channels.

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 (Ca(2+))(4)-CaM to a basic amphipathic helix in CaMKII releases auto-inhibition of the kinase.

A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation.

Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM.

Interdomain cooperativity of calmodulin bound to melittin preferentially increases calcium affinity of sites I and II.

Calmodulin (CaM) is the primary transducer of calcium fluxes in eukaryotic cells. Its two domains allosterically regulate myriad target proteins through calcium-linked association and conformational change. Many of these proteins have a basic amphipathic alpha-helix (BAA) motif that binds one or both CaM domains.

The NMDA receptor NR1 C1 region bound to calmodulin: structural insights into functional differences between homologous domains.

Calmodulin (CaM) regulates tetrameric N-methyl-D-aspartate receptors (NMDARs) by binding tightly to the C0 and C1 regions of its NR1 subunit. A crystal structure (2HQW; 1.96 A) of calcium-saturated CaM bound to NR1C1 (peptide spanning 875-898) showed that NR1 S890, whose phosphorylation regulates membrane localization, was solvent protected, whereas the endoplasmic reticulum retention motif was solvent exposed.

The neuronal voltage-dependent sodium channel type II IQ motif lowers the calcium affinity of the C-domain of calmodulin.

Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels.

Displacement of alpha-actinin from the NMDA receptor NR1 C0 domain By Ca2+/calmodulin promotes CaMKII binding.

Ca2+ influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor triggers activation and postsynaptic accumulation of Ca2+/calmodulin-dependent kinase II (CaMKII). CaMKII, calmodulin, and alpha-actinin directly bind to the short membrane proximal C0 domain of the C-terminal region of the NMDA receptor NR1 subunit. In a negative feedback loop, calmodulin mediates Ca2+-dependent inactivation of the NMDA receptor by displacing alpha-actinin from NR1 C0 upon Ca2+ influx.

HEXIM1 is a promiscuous double-stranded RNA-binding protein and interacts with RNAs in addition to 7SK in cultured cells.

P-TEFb regulates eukaryotic gene expression at the level of transcription elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein HEXIM1 or HEXIM2. In an effort to determine the minimal region of 7SK needed to interact with HEXIM1 in vitro, we found that an oligo comprised of nucleotides 10-48 sufficed. A bid to further narrow down the minimal region of 7SK led to a surprising finding that HEXIM1 binds to double-stranded RNA in a sequence-independent manner.