C-domain Of Calmodulin Research Articles

Neurogranin modulates the rate of association between calmodulin and target peptides

The best-known mode of action of calmodulin (CaM) is binding of Ca2+ to its N- and C-domains, followed by binding to target proteins. An underappreciated facet of this process is that CaM is typically bound to proteins at basal levels of free Ca2+, including the small, intrinsically disordered, neuronal IQ-motif proteins called PEP-19 and neurogranin (Ng). PEP-19 and Ng would not be effective competitive inhibitors of high-affinity Ca2+-dependent CaM targets at equilibrium because they bind to CaM with relatively low affinity, but they could influence the time course of CaM signaling by affecting the rate of association of CaM with high-affinity Ca2+-dependent targets. This mode of regulation may be domain specific because PEP-19 binds to the C-domain of CaM, whereas Ng binds to both N- and C-domains. In this report, we used a model CaM binding peptide (CKIIp) to characterize the preferred pathway of complex formation with Ca2+-CaM at low levels of free Ca2+ (0.25–1.5 μM), and how PEP-19 and Ng affect this process. We show that the dominant encounter complex involves association of CKIIp with the N-domain of CaM, even though the C-domain has a greater affinity for Ca2+. We also show that Ng greatly decreases the rate of association of Ca2+-CaM with CKIIp due to the relatively slow dissociation of Ng from CaM, and to interactions between the Gly-rich C-terminal region of Ng with the N-domain of CaM, which inhibits formation of the preferred encounter complex with CKIIp. These results provide the general mechanistic paradigms that binding CaM to targets can be driven by its N-domain, and that low-affinity regulators of CaM signaling have the potential to influence the rate of activation of high-affinity CaM targets and potentially affect the distribution of limited CaM among multiple targets during Ca2+ oscillations.

Energetics of Calmodulin Recognition of a Skeletal Muscle Ryanodine Receptor Site

In skeletal muscle, the calcium-binding protein calmodulin (CaM) modulates the opening and closing of the ryanodine receptor (RyR1), the main ion channel responsible for calcium release from the sarcoplasmic reticulum during excitation-contraction coupling. By weakly activating RyR1 at low (nM) calcium and inhibiting it at high (µM) calcium, CaM acts as a feedback regulator of calcium levels during muscle contraction. A short region on RyR1 (residues 3614-3640, RyR1p) serves as an anchor for CaM. We investigated the energetics of CaM-RyR1p recognition using Förster resonance energy transfer in auto-fluorescent biosensor constructs (YFP-RyRp-CFP). Using this system, we systematically explored the roles of individual RyR1 residues and each CaM domain at the interface, both in the presence and the absence of calcium. The interplay between the processes of calcium- and target-binding to CaM was probed by quantifying the energy of interaction between wild-type or mutated RyR1p sequences and CaM mutants having non-functional domain-specific Ca2+-binding sites. At low calcium, the interaction is weak (low µM), and mutation Trp3620Ala in the N-terminus of RyR1p (but not mutation Tyr3636Ala in the C-terminus) renders it non-specific. At high calcium, the interaction is at least three orders of magnitude stronger, and CaM C-domain binding to the N-terminal half of RyRp is the main mediator of recognition. A CaM C-domain Ca2+-binding mutant and/or Trp3620Ala RyR1p mutation significantly decrease the binding affinity, while a CaM N-domain Ca2+-binding mutant and/or Tyr3636Ala RyRp mutation have weaker effects. The primary role of CaM C-domain at the interface is maintained in the Tyr3636Ala RyRp complex, whereas Trp3620Ala mutation almost equalizes the energetic contributions of the two CaM domains. Future NMR studies of co-expressed CaM-RyR1p complexes will complement the thermodynamic data with atomic-resolution structural information.

Role of cardiac ryanodine receptor calmodulin-binding domains in mediating the action of arrhythmogenic calmodulin N-domain mutation N54I.

The Ca2+ -sensing protein calmodulin (CaM) inhibits cardiac ryanodine receptor (RyR2)-mediated Ca2+ release. CaM mutations associated with arrhythmias and sudden cardiac death have been shown to diminish CaM-dependent inhibition of RyR2, but the underlying mechanisms are not well understood. Nearly all arrhythmogenic CaM mutations identified are located in the C-domain of CaM and exert marked effects on Ca2+ binding to CaM and on the CaM C-domain interaction with the CaM-binding domain 2 (CaMBD2) in RyR2. Interestingly, the arrhythmogenic N-domain mutation CaM-N54I has little or no effect on Ca2+ binding to CaM or the CaM C-domain-RyR2 CaMBD2 interaction, unlike all CaM C-domain mutations. This suggests that CaM-N54I may diminish CaM-dependent RyR2 inhibition by affecting CaM N-domain interactions with RyR2 CaMBDs other than CaMBD2. To explore this possibility, we assessed the effects of deleting each of the four known CaMBDs in RyR2 (CaMBD1a, -1b, -2, or -3) on the CaM-dependent inhibition of RyR2-mediated Ca2+ release in HEK293 cells. We found that removing CaMBD1a, CaMBD1b, or CaMBD3 did not alter the effects of CaM-N54I or CaM-WT on RyR2 inhibition. On the other hand, deleting RyR2-CaMBD2 abolished the effects of both CaM-N54I and CaM-WT. Our results support that CaM-N54I causes aberrant RyR2 regulation via an uncharacterized CaMBD or less likely CaMBD2, and that RyR2 CaMBD2 is required for the actions of both N- and C-domain CaM mutations. Moreover, our results show that CaMBD1a is central to RyR2 regulation, but CaMBD1a, CaMBD1b, and CaMBD3 are not required for CaM-dependent inhibition of RyR2 in HEK293 cells.

Genetic Mosaicism in Calmodulinopathy.

CaM (calmodulin) mutations are associated with congenital arrhythmia susceptibility (calmodulinopathy) and are most often de novo. In this report, we sought to broaden the genotype-phenotype spectrum of calmodulinopathies with 2 novel calmodulin mutations and to investigate mosaicism in 2 affected families. CaM mutations were identified in 4 independent cases by DNA sequencing. Biochemical and electrophysiological studies were performed to determine functional consequences of each mutation. Genetic studies identified 2 novel CaM variants (CALM3-E141K in 2 cases; CALM1-E141V) and one previously reported CaM pathogenic variant (CALM3-D130G) among 4 probands with shared clinical features of prolonged QTc interval (range 505-725 ms) and documented ventricular arrhythmia. A fatal outcome occurred for 2 of the cases. The parents of all probands were asymptomatic with normal QTc duration. However, 2 of the families had multiple affected offspring or multiple occurrences of intrauterine fetal demise. The mother from the family with recurrent intrauterine fetal demise exhibited the CALM3-E141K mutant allele in 25% of next-generation sequencing reads indicating somatic mosaicism, whereas CALM3-D130G was present in 6% of captured molecules of the paternal DNA sample, also indicating mosaicism. Two novel mutations (E141K and E141V) impaired Ca2+ binding affinity to the C-domain of CaM. Human-induced pluripotent stem cell-derived cardiomyocytes overexpressing mutant or wild-type CaM showed that both mutants impaired Ca2+-dependent inactivation of L-type Ca2+ channels and prolonged action potential duration. We report 2 families with somatic mosaicism associated with arrhythmogenic calmodulinopathy, and demonstrate dysregulation of L-type Ca2+ channels by 2 novel CaM mutations affecting the same residue. Parental mosaicism should be suspected in families with unexplained fetal arrhythmia or fetal demise combined with a documented CaM mutation.

Interaction of NaV1.2 IQ Motif with Disease-Causing Mutants of Calmodulin

Calmodulin (CaM) binds constitutively to an IQ motif (residues 1901-1927, NaV1.2IQp) in the cytosolic C-terminal tail of the a-subunit of the neuronal voltage-gated sodium channel NaV1.2. Solution structures of CaM C-domain (CaMC) bound to NaV1.2IQp showed calcium binding reverses the orientation of (Ca2+)2-CaMC (2M5E.pdb) relative to apo CaMC (2KXW.pdb). Our structure of full-length apo human CaM bound to human NaV1.2IQp showed that only CaMc interacts with NaV1.2IQp. Thermodynamic and NMR analysis of the CaM-NaV1.2IQp complex showed no evidence for CaMN-NaV1.2IQp interactions, and indicated nearly identical affinity for calcium binding to the 4 EF-hand sites of CaM. Residues in NaV1.2IQp had different chemical environments when bound to apo and (Ca2+)4-CaM, consistent with the calcium-trigged change observed in the (Ca2+)2-CaMC-NaV1.2IQp interface (2M5E.pdb). Apo human CaM carrying cardiomyopathy-causing mutations [N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)] all bound tightly to NaV1.2IQp. However, mutations in CaMC inhibited Ca2+-mediated conformational changes of CaM bound to NaV1.2IQp. This suggests that high cytosolic Ca2+ levels would not be sufficient to trigger reversal of the orientation of these CaMC mutants. The effects of other recently described disease-causing CaM mutations are being investigated. NIH R01 GM57001 (MAS), NIH T32 NS045549 (RM), UI CCOM FUTURE in Biomedicine (AMK)

Ca2+-dependent calmodulin binding to cardiac ryanodine receptor (RyR2) calmodulin-binding domains.

The Ca2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca2+-dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhythmias. However, the molecular details of the CaM–RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca2+-dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca2+ concentrations above 0.2 µM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca2+ saturated under resting Ca2+ conditions. Conversely, the N-domain senses Ca2+ exactly in the transition from resting to activating Ca2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca2+ during Ca2+ oscillations, while the CaM N-domain functions as a dynamic Ca2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.

Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels NaV1.1, NaV1.4 and NaV1.7.

Human voltage-gated sodium (NaV) 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. NaV pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all NaV isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of NaV1.2, NaV1.5, and NaV1.6 showed that CaM binding was mediated by the C-domain of CaM (CaMC), while the N-domain (CaMN) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other NaV isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of NaV1.1, NaV1.4, and NaV1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaMC; resonances of CaMN were identical to free CaM. Thus, CaMC residues contribute to the interface with the IQ motif, while CaMN is available to interact elsewhere on the channel.

Direct visualization of interaction between calmodulin and connexin45.

Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+ signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins; however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here, we report the first direct interaction of CaM with Cx45 (connexin45) of γ-family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer. The interaction between CaM and Cx45 in cells is strongly dependent on intracellular Ca2+ concentration and can be blocked by the CaM inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7). We further reveal a CaM-binding site at the cytosolic loop (residues 164-186) of Cx45 using a peptide model. The strong binding (Kd ∼ 5 nM) observed between CaM and Cx45 peptide, monitored by fluorescence-labeled CaM, is found to be Ca2+-dependent. Furthermore, high-resolution nuclear magnetic resonance spectroscopy reveals that CaM and Cx45 peptide binding leads to global chemical shift changes of 15N-labeled CaM, but does not alter the size of the structure. Observations involving both N- and C-domains of CaM to interact with the Cx45 peptide differ from the embraced interaction with Cx50 from another connexin family. Such interaction further increases Ca2+ sensitivity of CaM, especially at the N-terminal domain. Results of the present study suggest that both helicity and the interaction mode of the cytosolic loop are likely to contribute to CaM's modulation of connexins.

Backbone resonance assignments of complexes of human voltage-dependent sodium channel NaV1.2 IQ motif peptide bound to apo calmodulin and to the C-domain fragment of apo calmodulin.

Human voltage-gated sodium channel NaV1.2 has a single pore-forming α-subunit and two transmembrane β-subunits. Expressed primarily in the brain, NaV1.2 is critical for initiation and propagation of action potentials. Milliseconds after the pore opens, sodium influx is terminated by inactivation processes mediated by regulatory proteins including calmodulin (CaM). Both calcium-free (apo) CaM and calcium-saturated CaM bind tightly to an IQ motif in the C-terminal tail of the α-subunit. Our thermodynamic studies and solution structure (2KXW) of a C-domain fragment of apo 13C,15N- CaM (CaMC) bound to an unlabeled peptide with the sequence of rat NaV1.2 IQ motif showed that apo CaMC (a) was necessary and sufficient for binding, and (b) bound more favorably than calcium-saturated CaMC. However, we could not monitor the NaV1.2 residues directly, and no structure of full-length CaM (including the N-domain of CaM (CaMN)) was determined. To distinguish contributions of CaMN and CaMC, we used solution NMR spectroscopy to assign the backbone resonances of a complex containing a 13C,15N-labeled peptide with the sequence of human NaV1.2 IQ motif (NaV1.2IQp) bound to apo 13C,15N-CaM or apo 13C,15N-CaMC. Comparing the assignments of apo CaM in complex with NaV1.2IQp to those of free apo CaM showed that residues within CaMC were significantly perturbed, while residues within CaMN were essentially unchanged. The chemical shifts of residues in NaV1.2IQp and in the C-domain of CaM were nearly identical regardless of whether CaMN was covalently linked to CaMC. This suggests that CaMN does not influence apo CaM binding to NaV1.2IQp.

Backbone and side-chain resonance assignments of (Ca2+)4-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 (CaNA) 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. The N- and C-domains of CaM (CaMN and CaMC) recognize a CaM-binding domain (CaMBD) within an intrinsically disordered region of CaNA that precedes the auto-inhibitory domain (AID) of CaNA. Here we present nearly complete 1H, 13C, and 15N resonance assignments of (Ca2+)4-CaM bound to a peptide containing the CaMBD sequence in the beta isoform of CaNA (βCaNA-CaMBDp). Its secondary structure elements predicted from the assigned chemical shifts were in good agreement with those observed in the high-resolution structures of (Ca2+)4-CaM bound to CaMBDs of multiple enzymes. Based on the reported literature, the CaMBD of the α isoform of CaNA can bind to CaM in two opposing orientations which may influence the regulatory function of CaM. Because a high resolution structure of (Ca2+)4-CaM bound to βCaNA-CaMBDp has not been reported, our studies serve as a starting point for determining the solution structure of this complex. This will demonstrate the preferred orientation of (Ca2+)4-CaM on the CaMBD as well as the orientations of CaMN and CaMC relative to each other and to the AID of βCaNA.

Opposing Intermolecular Tuning of Ca2+ Affinity for Calmodulin by Neurogranin and CaMKII Peptides

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca2+) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca2+/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca2+ signaling pathways in neurons. It was shown experimentally that Ca2+/CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca2+-free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng13–49), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng13–49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng13–49 structures by matching the changes in the chemical shifts of CaM upon Ng13–49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski’s equality. We discovered the molecular underpinnings of lowered affinity of Ca2+ for CaM in the presence of Ng13–49 by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca2+ binding loops particularly at C-domain of CaM, enabling Ca2+ release. In contrast, CaMKII peptide increases Ca2+ affinity for the C-domain of CaM by stabilizing the two Ca2+ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng13–49 and holoCaM-CaMKII delineates the importance of CaM’s progressive mechanism of target binding on its Ca2+ binding affinities.

Opposing Intermolecular Tuning of Ca2+ Affinity for Calmodulin by Target Peptides

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca2+) by integrating coarse-grained models and all-atomistic simulations with non-equilibrium physics. We focused on binding between CaM and two specific targets, Ca2+/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca2+ signaling pathways in neurons. It was shown experimentally that Ca2+/CaM binds to the CaMKII peptide with higher affinity than the Ng peptide. The binding of CaMKII peptide to CaM in return increases the Ca2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide that binds to Ca2+-free CaM or Ca2+/CaM with similar binding affinity. Unlike CaM-CaMKII peptide that renders solved crystal structures, the structural description of CaM-Ng peptide is unknown due to low binding affinity, therefore, we computationally generated an ensemble of CaM-Ng peptide structures by matching the changes in the chemical shifts of CaM upon Ng peptide binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca2+ for CaM in the presence of Ng by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca2+ binding loops particularly at C-domain of CaM, enabling Ca2+ release. In contrast, CaMKII increases Ca2+ affinity for the C-domain of CaM by stabilizing the two Ca2+ binding loops.

PEP-19 modulates calcium binding to calmodulin by electrostatic steering.

PEP-19 is a small protein that increases the rates of Ca2+ binding to the C-domain of calmodulin (CaM) by an unknown mechanism. Although an IQ motif promotes binding to CaM, an acidic sequence in PEP-19 is required to modulate Ca2+ binding and to sensitize HeLa cells to ATP-induced Ca2+ release. Here, we report the NMR solution structure of a complex between PEP-19 and the C-domain of apo CaM. The acidic sequence of PEP-19 associates between helices E and F of CaM via hydrophobic interactions. This allows the acidic side chains in PEP-19 to extend toward the solvent and form a negatively charged surface that resembles a catcher's mitt near Ca2+ binding loop III of CaM. The topology and gradients of negative electrostatic surface potential support a mechanism by which PEP-19 increases the rate of Ca2+ binding to the C-domain of CaM by ‘catching' and electrostatically steering Ca2+ to site III.

Structural Differences in Calmodulin Bound to Voltagate-Gated Sodium Channel IQ Motifs

Voltage-gated sodium channels (NaV) control the influx of Na+ and the rising phase of action potentials in excitable cells. They bind calmodulin (CaM) at an IQ motif in their C-terminal domain (CTD), but calcium-mediated regulation is poorly understood. Five high-resolution structures of apo (calcium-depleted) CaM bound to the IQ motif of NaV1.2, NaV1.5 and NaV1.6 show the NaV primarily contacts the C-domain of CaM (CaMC), which is in a “semi-open” tertiary conformation. In these structures, the IQ motif adopts the same orientation relative to apo CaMC. In two structures (4JPZ/Xray and 2M5E/NMR) of Ca2+-CaM bound to IQ-containing fragments of NaV1.2, Ca2+-CaM primarily makes contacts with residues 1905-1921. However, the orientation of the IQ motif relative to CaMC in 2M5E is opposite to that of 4JPZ. In 4JPZ (X-ray), the NaV1.2 CTD (residues 1777-1937, including the EF-like domain and IQ region), binds “semi-open” CaMC, whereas in 2M5E (NMR), CaMC is “open” when bound to NaV1.2 residues 1901-1927 (I1912 and Q1913 are the IQ residues). These extremely different CaM-IQ complexes may both be populated in the cell. To explore how the NaV1.2 sequence outside the IQ motif may determine these differences, we used solution (NMR) studies to compare full-length human Ca2+-CaM bound to two NaV1.2 fragments - residues 1777-1937 and 1901-1927. In both complexes, Ca2+-CaMC adopted the “open” conformation (observed in 2M5E and other Ca2+-CaMstructures), suggesting that differences in the sequences flanking the NaV1.2 IQ motif are not sufficient to explain the differences between 4JPZ and 2M5E. Future structural studies will explore how different solution conditions used in the 4JPZ (X-ray) and 2M5E (NMR) structures may select for different conformations of CaM bound to IQ motifs. Supported by NIH R01 GM57001.

Conformational frustration in calmodulin-target recognition.

Calmodulin (CaM) is a primary calcium (Ca(2+) )-signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM-binding domains from two Ca(2+) -CaM-dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM-CaMKII complex than in the CaM-CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C-domain of CaM that need to be broken before the formation of intermolecular contacts with the N-domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations.

NMR Structure Reveals Novel Interactions between Intrinsically Disordered PEP-19 and Calmodulin

Background: PEP-19 has no known intrinsic activity other than binding predominately to the C-domain of calmodulin (C-CaM), yet it is implicated in numerous cellular processes. We showed that an acidic sequence in PEP-19 is required to greatly increase the rates of Ca2+ binding to C-CaM. Importantly, the acidic sequence is also required for PEP-19 to sensitize HeLa cells to ATP-induced Ca2+ release. Goal: The goal of the current study was to determine the high-resolution NMR solution structure of the PEP-19/apo C-CaM complex.Results: Apo C-CaM adopts a semi-open conformation when bound to PEP-19, with the helices E and F of Ca2+ binding site III showing the greatest change in angle relative to free apo C-CaM. The conformation of Ca2+ binding loop III is similar to that in free apo C-CaM, but loop IV adopts a different conformation with increased conformational exchange when bound to PEP-19. Residues 1-29 in PEP-19 remain disordered when bound to C-CaM, and are thus accessible for potential interactions with other proteins. The IQ motif in PEP-19 adopts a well-defined alpha helix that binds to a hydrophobic groove in apo C-CaM. The C-terminal part of the acidic sequence is alpha helical, but the N-terminal portion (aa 28-36) forms loop and coil structures that are stabilized by interactions between Ile32 and Met34 in PEP-19 and hydrophobic residues in Ca2+ binding site III of C-CaM. This allows acid side chains in PEP-19 to extend toward the solvent to greatly increase negative charge density near site III of C-CaM.Conclusions: The structure suggests that the acidic sequence in PEP-19 modulates Ca2+ binding to site III of C-CaM by direct interactions and/or electrostatic steering of Ca2+, but that allosteric effects increase conformational exchange to modulate Ca2+ binding to site IV.

Thermodynamic and Structural Analysis of Calmodulin Interaction with the Skeletal Muscle Ryanodine Receptor

In skeletal muscle, the calcium sensor calmodulin (CaM) plays a key role in excitation-contraction coupling, the process of translating neuronal stimuli into mechanical contraction of muscle, by regulating the opening and closing of the calcium channel ryanodine receptor (RyR1). By interacting with this channel differently at high and low calcium, CaM acts as a feedback regulator of calcium levels during muscle contraction: at low calcium, CaM weakly activates RyR1, while calcium-CaM inhibits it. We are investigating the interaction between CaM and its established binding site on RyR1 (CaMBD, residues 3614-3640) using a multi-faceted approach combining biophysical (fluorescence spectroscopy) and structural (solution NMR) methodologies. Forster resonance energy transfer (FRET) experiments in an auto-fluorescent biosensor construct (YFP-CaMBD-CFP) enabled us to determine Gibbs free energies of binding in the absence and the presence of calcium. Using this system, we systematically explored the thermodynamics of molecular recognition between the two biomolecules in high and low calcium environments, as well as the roles played by individual RyR1 residues and each CaM lobe at the interface. To gain additional insights into the interplay between the processes of calcium- and target-binding to CaM, we analyzed the interaction between wild-type or mutated RyR1 CaMBD sequences and CaM mutants in which the calcium-binding sites in one domain had been rendered non-functional. Overall, these experiments show that CaM C-domain binding to molecular determinants in the N-terminus of RyR1 CAMBD dominates the interaction, both in the presence and the absence of calcium. However, the interaction is three orders of magnitude stronger at high calcium levels. To obtain residue-specific information of the binding interface, we are currently undertaking NMR studies of isotopically labeled CaM (wild-type and calcium-binding mutants) in the absence and presence of wild-type and mutated RyR1 sequences.

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 (Ca2+)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 hRyR11975–1999 or hRyR13614-3643. Both CaM and its C-domain associated in a calcium-independent manner with hRyR13614–3643 while N-domain required calcium and bound ~250-fold more weakly. Association with hRyR111975–1999 was weak. Both hRyR1 peptides increased the calcium-binding affinity of both CaM domains, while maintaining differences between them. These energetics support the CaM C-domain association with hRyR13614–3643 at low calcium, positioning CaM to respond to calcium efflux. However, the CaM N-domain affinity for hRyR11975–1999 alone was insufficient to support CaM bridging adjacent RyR1 subunits. Other proteins or elements of the hRyR1 structure must contribute to the energetics of CaM-mediated regulation.

Neurogranin Alters the Structure and Calcium Binding Properties of Calmodulin

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 Ca(2+) 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 Ca(2+) binding to the C-terminal domain of CaM with an associated increase in the Ca(2+) dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca(2+) binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca(2+)-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 Ca(2+) 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 Ca(2+)-bound CaM and that although Ca(2+) binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca(2+) binding to the C-terminal lobe of CaM.

An Acidic Sequence in Neurogranin is Required to Modulate Ca2+ Binding to Calmodulin

Background: Neurogranin (Ng) and PEP-19 are small proteins with no known intrinsic activity other than binding to calmodulin (CaM) via their IQ motifs, yet they have been implicated in numerous normal and pathological processes. We showed that PEP-19 is intrinsically disordered, and that an acidic sequence adjacent to its IQ motif is required for PEP-19 to modulate Ca2+ binding to CaM, and to sensitize HeLa cells to ATP-induced Ca2+ release. Goals: Ng has an acidic sequence, but with significantly different composition than PEP-19. Thus, the goals of the current study were to determine: 1) If the acidic sequence in Ng is required to modulate Ca2+ binding to CaM; and 2) Use NMR to compare the effects of Ng derivatives on the conformation of CaM. Results: Ng greatly increases the Ca2+ koff at the C-domain of CaM, but has little effect on the kon, thereby decreasing Ca2+ binding affinity. The peptide Ng(29-49), which includes only the consensus IQ motif does not increase the Ca2+ koff, but Ng(13-49), which includes the acidic region of Ng mimics the effect of intact Ng on Ca2+ binding to CaM. The 1H 15N HSQC spectra of Ng show it to be an intrinsically disordered protein. Effects of Ng protein and peptides on the NMR spectra of CaM are consistent with their relative effects on Ca2+ binding. Also, effects of Ng (29-49) on amide chemical shift perturbations and backbone dynamic properties of CaM are different from Ng or Ng(13-49), especially in the apo state. Conclusions: Modulating Ca2+ binding to CaM relies on the acidic region of both PEP-19 and Ng. Tuning Ca2+ mobilization pathways by PEP-19 and Ng expands the biological significance of these intrinsically disordered regulators of CaM signaling.