EF-hands Of Calmodulin Research Articles

The Crystal Structure of Calmodulin Bound to the Cardiac Ryanodine Receptor (RyR2) at Residues Phe4246-Val4271 Reveals a Fifth Calcium Binding Site.

Calmodulin (CaM) regulates the activity of a Ca2+ channel known as the cardiac ryanodine receptor (RyR2), which facilitates the release of Ca2+ from the sarcoplasmic reticulum during excitation-contraction coupling in cardiomyocytes. Mutations that disrupt this CaM-dependent channel inactivation result in cardiac arrhythmias. RyR2 contains three different CaM binding sites: CaMBD1 (residues 1940-1965), CaMBD2 (residues 3580-3611), and CaMBD3 (residues 4246-4275). Here, we report a crystal structure of Ca2+-bound CaM bound to RyR2 CaMBD3. The structure reveals Ca2+ bound to the four EF-hands of CaM as well as a fifth Ca2+ bound to CaM in the interdomain linker region involving Asp81 and Glu85. The CaM mutant E85A abolishes the binding of the fifth Ca2+ and weakens the binding of CaMBD3 to Ca2+-bound CaM. Thus, the binding of the fifth Ca2+ is important for stabilizing the complex in solution and is not a crystalline artifact. The CaMBD3 peptide in the complex adopts an α-helix (between Phe4246 and Val4271) that interacts with both lobes of CaM. Hydrophobic residues in the CaMBD3 helix (Leu4255 and Leu4259) form intermolecular contacts with the CaM N-lobe, and the CaMBD3 mutations (L4255A and L4259A) each weaken the binding of CaM to RyR2. Aromatic residues on the opposite side of the CaMBD3 helix (Phe4246 and Tyr4250) interact with the CaM C-lobe, but the mutants (F4246A and Y4250A) have no detectable effect on CaM binding in solution. We suggest that the binding of CaM to CaMBD3 and the binding of a fifth Ca2+ to CaM may contribute to the regulation of RyR2 channel function.

Conformational Structural Alteration of NCS1 Protein Through Pb2+and Mn2+ Association

NCS1 (Frequenin) is a member of neuronal calcium sensor (NCS) family. This globular protein contains four EF-hand motifs and an amino-terminal myristylation sequence. Pb2+has been widely viewed as a health concern for its anthropogenic toxicity. It has been observed that Pb2+ modulates interactions between calcium-binding protein calmodulin (CaM) and its binding partners through binding in the EF-hands of CaM and it may play a significant role in Pb2+-induced neurotoxicity. Mn is a highly abundant metal in nature which has been found in human brain and associated with the several diseases like multiple sclerosis, Alzheimer's and Parkinson's disease and is considered as potential toxic element. Previous studies from our group showed Pb2+ and Cd2+ binding to DREAM protein that can contribute to Pb2+/Cd2+ induced neurotoxicity. Here we showed that Pb2+ binds to EF-hands in apo-NCS1with a high affinity (Kd = 7.0±1.5 nM) whereas Cd2+ did not show any binding with NCS1. For the first time we investigated the Mn2+ association to NCS1 protein. This cation binds with an apparent equilibrium dissociation constant of 340±67 µM. Pb2+ and Mn2+binding trigger secondary and tertiary structural rearrangements of NCS1 that are analogous to those observed for Ca2+ association. In addition, Pb2+ as well as Mn2+ promote NCS1 interaction with hydrophobic molecule 1,8-ANS. Interestingly,Ca2+NCS1:1,8-ANS complex exhibits a co-operative binding of Pb2+ with Kd = 103±23 µM whereas Mn2+ showed apparent Kd value 924±185 µM with Ca2+NCS1:1,8-ANS complex. These results suggest allosteric interactions between the 1,8-ANS binding site and Pb2+ binding EF-hands. Data provided here points towards ability of EF-hands in NCS-1 to interact with various metals and a possible role of NCS1 in Pb2+/Mn2+ neurotoxicity.

Coordination to lanthanide ions distorts binding site conformation in calmodulin

The Ca2+-sensing protein calmodulin (CaM) is a popular model of biological ion binding since it is both experimentally tractable and essential to survival in all eukaryotic cells. CaM modulates hundreds of target proteins and is sensitive to complex patterns of Ca2+ exposure, indicating that it functions as a sophisticated dynamic transducer rather than a simple on/off switch. Many details of this transduction function are not well understood. Fourier transform infrared (FTIR) spectroscopy, ultrafast 2D infrared (2D IR) spectroscopy, and electronic structure calculations were used to probe interactions between bound metal ions (Ca2+ and several trivalent lanthanide ions) and the carboxylate groups in CaM's EF-hand ion-coordinating sites. Since Tb3+ is commonly used as a luminescent Ca2+ analog in studies of protein-ion binding, it is important to characterize distinctions between the coordination of Ca2+ and the lanthanides in CaM. Although functional assays indicate that Tb3+ fully activates many Ca2+-dependent proteins, our FTIR spectra indicate that Tb3+, La3+, and Lu3+ disrupt the bidentate coordination geometry characteristic of the CaM binding sites' strongly conserved position 12 glutamate residue. The 2D IR spectra indicate that, relative to the Ca2+-bound form, lanthanide-bound CaM exhibits greater conformational flexibility and larger structural fluctuations within its binding sites. Time-dependent 2D IR lineshapes indicate that binding sites in Ca2+-CaM occupy well-defined configurations, whereas binding sites in lanthanide-bound-CaM are more disordered. Overall, the results show that binding to lanthanide ions significantly alters the conformation and dynamics of CaM's binding sites.

Inside Cover: Nanosecond Dynamics of Calmodulin and Ribosome‐Bound Nascent Chains Studied by Time‐Resolved Fluorescence Anisotropy (ChemBioChem 7/2014)

The inside cover picture shows a side view of the growing, FlAsH-labeled calmodulin (CaM) chain bound on the ribosome. Bottom right is a top view of labeled CaM after synthesis. Yellow/orange: ribosomal subunits, green/red: first/second EF hands of CaM, blue: C-terminal half of CaM. For details see the paper by J. Fitter et al. on p. 977 ff.

Fine Tuning of Calmodulin's Affinity for Ca2+ by Target Proteins

Calmodulin (CaM) is a prototypical and versatile Ca2+ sensor. CaM is present in all eukaryotic cells, mediating Ca2+-dependent signaling. Upon binding Ca2+, CaM changes its conformation to form complexes with a diverse array of target proteins. Despite a wealth of knowledge on CaM, little is known on how target proteins regulate CaM's ability to bind Ca2+. Recently we identified a novel splice variant (KCNN2-b) of KCNN channels which shows different Ca2+ sensitivity compared to the original KCNN2-a. KCNN2-b has three additional amino acid residues in the CaM binding domain (CaMBD). We take advantage of the CaMBDs from these splice variants to investigate their impact on CaM. Crystal structures show that interactions with these KCNN2 splice variants force CaM to adopt different conformations and thus change its affinity for Ca2+ at its C-lobe. All four EF-hands of CaM in the CaM-CaMBD2-b complex are occupied by Ca2+ ions, whereas in the CaM-CaMBD2-a complex only EF-hands at CaM N-lobe are loaded. Experiments with mutant CaMs support this difference in Ca2+ binding stoichiometry. Mutations of the EF hands at CaM N-lobe (CaM12) almost abolished the interaction with both CaMBDs. The mutant CaM unable to bind Ca2+ at C-lobe (CaM34) interacts with CaMBD2-a substantially, with a similar amplitude compared to wild type CaM. The thermodynamic profile of CaM34-CaMBD2-b, however, is distinct from that of the wild type CaM-CaMBD2-b. This difference in metal ion binding stoichiometry is also proven in solution by Tb3+ Fluorescence measurements. We further manipulated the three residues insertion by shortening it to two residues (CaMBD2-AlaAla) or one residue (CaMBD2-Ala). Different metal ion binding stoichiometry and Ca2+ sensitivity was observed between CaM complexes with these two mutant CaMBDs, which furthers our understanding on the fine-tuning of CaM's affinity for Ca2+ by target proteins.

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. The crystal structure of CaM bound to CaMKIIp, a peptide representing the CaM-binding domain (CaMBD) of CaMKII, shows an antiparallel interface: the C-domain of CaM primarily contacts the N-terminal half of the CaMBD. The two domains of calcium-saturated CaM are believed to play distinct roles in releasing auto-inhibition. To investigate the underlying mechanism of activation, calcium-dependent titrations of isolated domains of CaM binding to CaMKIIp were monitored using fluorescence anisotropy. The binding affinity of CaMKIIp for the domains of CaM increased upon saturation with calcium, with the C-domain having a 35-fold greater affinity than the N-domain. Because the interdomain linker of CaM regulates calcium-binding affinity and contribute to conformational change, the role of each CaM domain was explored further by investigating effects of CaMKIIp on site-knockout mutants affecting the calcium-binding sites of a single domain. Investigation of the thermodynamic linkage between saturation of individual calcium-binding sites and CaM-domain binding to CaMKIIp showed that calcium binding to Sites III and IV was sufficient to recapitulate the behavior of (Ca(2+))(4)-CaM. The magnitude of favorable interdomain cooperativity varied depending on which of the four calcium-binding sites were mutated, emphasizing differential regulatory roles for the domains of CaM, despite the high degree of homology among the four EF-hands of CaM.

Conformation and Structural Transitions in the EF-Hands of Calmodulin

Calcium plays a key role in cellular signal transduction. Calmodulin, a protein binding four calcium ions, is found in all eukaryotic cells and is believed to activate such processes. The calcium binding loop found in this protein, the canonical EF-hand, is also found in a large number of other proteins such as troponins, parvalbumins, calbindins etc. Earlier analysis of the amino acid sequences of these proteins with a view of understanding evolution of protein families and signaling mechanisms have provided extensive evidence for a characteristic double gene duplication event in this family of proteins. These analyses have been extended here to the three dimensional structures and the biophysical properties of the sequence segments of calmodulin EF-hands. The clear evolutionary history that shows up in sequences is not reflected as clearly in the conformation of individual EF-hands, which may be a consequence of the much higher conservation pressure on the structure. Some evidence for the proposed gene duplication is implicit in the apo-holo structural transitions of the EF- hands. The profile of amino acid properties that might be significant for calcium binding, however, clearly reflects the gene duplication. These profiles might also provide insightful information on the calcium affinity of the EF-hand motifs and the nature of amino acid residues that constitute them.

Cellular Uptake and in Situ Binding of a Peptide Agonist for Calmodulin

We have used the method of inverted hydropathy to develop peptides that interact with EF hands of calmodulin (CaM). Previously we have shown these peptides specifically interact with their desired target in a productive manner, in that they activated CaM in the absence of Ca2+. Therefore, we sought to determine whether these peptides would enter cells, remain intact, and interact with CaM in the interior of the cell. Using several techniques we have demonstrated cellular uptake, stability, and an intracellular interaction with CaM with fluorescein-labeled and radiolabeled peptides in Jurkat T cells. The results suggest that these peptides may be useful in the study and the manipulation of Ca2+-mediated pathways in cells.

A new type of Ca(2+) channel blocker that targets Ca(2+) sensors and prevents Ca(2+)-mediated apoptosis.

Calmodulin (CaM), as well as other Ca(2+) binding motifs (i.e., EF hands), have been demonstrated to be Ca(2+) sensors for several ion channel types, usually resulting in an inactivation in a negative feedback manner. This provides a novel target for the regulation of such channels. We have designed peptides that interact with EF hands of CaM in a specific and productive manner. Here we have examined whether these peptides block certain Ca(2+)-permeant channels and inhibit biological activity that is dependent on the influx of Ca(2+). We found that these peptides are able to enter the cell and directly, as well as indirectly (through CaM), block the activity of glutamate receptor channels in cultured neocortical neurons and a nonselective cation channel in Jurkat T cells that is activated by HIV-1 gp120. As a consequence, apoptosis mediated by an influx of Ca(2+) through these channels was also dose-dependently inhibited by these novel peptides. Thus, this new type of Ca(2+) channel blocker may have utility in controlling apoptosis due to HIV infection or neuronal loss due to ischemia.

A new type of Ca2+channel blocker that targets Ca2+sensors and prevents Ca2+‐mediated apoptosis

Calmodulin (CaM), as well as other Ca2+ binding motifs (i.e., EF hands), have been demonstrated to be Ca2+ sensors for several ion channel types, usually resulting in an inactivation in a negative feedback manner. This provides a novel target for the regulation of such channels. We have designed peptides that interact with EF hands of CaM in a specific and productive manner. Here we have examined whether these peptides block certain Ca2+-permeant channels and inhibit biological activity that is dependent on the influx of Ca2+. We found that these peptides are able to enter the cell and directly, as well as indirectly (through CaM), block the activity of glutamate receptor channels in cultured neocortical neurons and a nonselective cation channel in Jurkat T cells that is activated by HIV-1 gp120. As a consequence, apoptosis mediated by an influx of Ca2+ through these channels was also dose-dependently inhibited by these novel peptides. Thus, this new type of Ca2+ channel blocker may have utility in contro...

Molecular cloning of a novel Ca2+-binding protein that is induced by NaCl stress.

Plant responses to high salt stress have been studied for several decades. However, the molecular mechanisms underlying these responses still elude us. In order to understand better the molecular mechanism related to NaCl stress in plants, we initiated the cloning of a large number of NaCl-induced genes in Arabidopsis. Here, we report the cloning of a cDNA encoding a novel Ca2+-binding protein, named AtCP1, which shares sequence similarities with calmodulins. AtCP1 exhibits, in particular, a high degree of amino acid sequence homology to the Ca2+-binding loops of the EF hands of calmodulin. However, unlike calmodulin, AtCP1 appears to have only three Ca2+-binding loops. We examined Ca2+ binding of the protein by a Ca2+-dependent electrophoretic mobility shift assay. A recombinant AtCP1 protein that was expressed in Escherichia coli did show a Ca2+-dependent electrophoretic mobility shift. To gain insight into the expression of the AtCP1 gene, northern blot analysis was carried out. The AtCP1 gene had a tissue-specific expression pattern: high levels of expression in flower and root tissues and nearly undetectable levels in leaves and siliques. Also, the expression of the AtCP1 gene was induced by NaCl treatment but not by ABA treatment. Finally, subcellular localization experiments using an AtCP1:smGFP fusion gene in soybean suspension culture cells and tobacco leaf protoplasts indicate that AtCP1 is most likely a cytosolic protein.

Localization of Unique Functional Determinants in the Calmodulin Lobes to Individual EF Hands

We have investigated the functional interchangeability of EF hands I and III or II and IV, which occupy structurally analogous positions in the native I-II and III-IV EF hand pairs of calmodulin. Our approach was to functionally characterize four engineered proteins, made by replacing in turn each EF hand in one pair by a duplicate of its structural analog in the other. In this way functional determinants we define as unique were localized to the component EF hands in each pair. Replacement of EF hand I by III reduces calmodulin-dependent activation of cerebellar nitric oxide synthase activity by 50%. Replacement of EF hand IV by II reduces by 60% activation of skeletal muscle myosin light chain kinase activity. There appear to be no major unique determinants for activation of these enzyme activities in the other EF hands. Replacement of EF hand III by I or IV by II reduces by 50-80% activation of smooth muscle myosin light chain kinase activity, and replacement of EF hand I by III or II by IV reduces by 90% activation of this enzyme activity. Thus, calmodulin-dependent activation of each of the enzyme activities examined, even the closely related kinases, is dependent upon a distinct pattern of unique determinants in the four EF hands of calmodulin. All the engineered proteins examined bind four Ca2+ ions with high affinity. Comparison of the Ca2+-binding properties of native and engineered CaMs indicates that the Ca2+-binding affinity of an engineered I-IV EF hand pair and a native I-II pair are similar, but an engineered III-II EF hand pair is intermediate in affinity to the native III-IV and I-II pairs, minimally suggesting that EF hands I and III contain unique determinants for the formation and function of EF hand pairs. The residues directly coordinating Ca2+ ion appear to play little or no role in establishing the different Ca2+-binding properties of the EF hand pairs in calmodulin.

Mutational effects on the cooperativity of Ca2+ binding in calmodulin.

The importance of the aspartate ligand in the +Y Ca2+ coordinating position of two EF-hands of calmodulin has been investigated. Synthetic calmodulin genes were used to produce engineered proteins with the wild-type bovine sequence as well as with aspartate 58 in Ca(2+)-binding site II and/or aspartate 95 in site III changed to asparagine. The macroscopic Ca(2+)-binding constants of the intact calmodulins and of tryptic fragments comprising the N- and C-terminal domains were determined from titrations with Ca2+ in the presence of 5,5'-Br2BAPTA. Substitution of aspartate by asparagine in Ca(2+)-binding site II led to a slight increase in the total free energy change on Ca2+ binding, and the cooperativity of Ca2+ binding to the N-terminal sites was substantially increased. The change from aspartate to asparagine in site III decreased the Ca2+ affinity and also appeared to decrease the positive cooperativity between the sites in the C-terminal domain. Thus, identical mutations in sites II and III were found to result in opposite effects. The data imply that involvement of liganding side chains in interactions other than direct calcium attraction and calcium coordination is of considerable importance for the Ca(2+)-binding process, particularly for the cooperativity.