Calcium-free Calmodulin Research Articles

Dynamic Interactions of Plant CNGC Subunits and Calmodulins Drive Oscillatory Ca2+ Channel Activities

Calcium is a universal signal in all eukaryotes, but themechanism for encoding calcium signatures remains largely unknown. Calcium oscillations control pollen tube growth and fertilization in flowering plants, serving as a model for dissecting the molecular machines that mediate calcium fluctuations. We report that pollen-tube-specific cyclic nucleotide-gated channels (CNGC18, CNGC8, and CNGC7) together with calmodulin 2 (CaM2) constitute a molecular switch that either opens or closes the calcium channel depending on cellular calcium levels. Under low calcium, calcium-free calmodulin 2 (Apo-CaM2) interacts with CNGC18-CNGC8 complex, leading to activation of the influx channel and consequently increasing cytosolic calcium levels. Calcium-bound CaM2 dissociates from CNGC18/8 heterotetramer, closing the channel and initiating a downturn of cellular calcium levels. We further reconstituted thecalcium oscillator in HEK293 cells, supporting the model that Ca2+-CaM-dependent regulation of CNGC channel activity provides an auto-regulatory feedback mechanism for calcium oscillations during pollen tube growth.

A structural comparison of ‘real’ and ‘model’ calmodulin clarified allosteric interactions regulating domain motion

Calmodulin (CaM) is a multifunctional calcium-binding protein, which regulates various biochemical processes. CaM acts via structural changes and complex forming with its target enzymes. CaM has two globular domains (N-lobe and C-lobe) connected by a long linker region. Upon calcium binding, the N-lobe and C-lobe undergo local conformational changes, after that, entire CaM wraps the target enzyme through a large conformational change. However, the regulation mechanism, such as allosteric interactions regulating the conformational changes, is still unclear. In order to clarify the allosteric interactions, in this study, experimentally obtained ‘real’ structures are compared to ‘model’ structures lacking the allosteric interactions. As the allosteric interactions would be absent in calcium-free CaM (apo-CaM), allostery-eliminated calcium-bound CaM (holo-CaM) models were constructed by combining the apo-CaM’s linker and the holo-CaM’s N- and C-lobe. Before the comparison, the ‘real’ and ‘model’ structures were clustered and cluster–cluster relationship was determined by a principal component analysis. The structures were compared based on the relationship, then, a distance map and a contact probability analysis clarified that the inter-domain motion is regulated by several groups of inter-domain contacting residue pairs. The analyses suggested that these residues cause inter-domain translation and rotation, and as a consequence, the motion encourage structural diversity. The resultant diversity would contribute to the functional versatility of CaM.

Exploring Kinetic Scenarios for Allosteric Transitions of Calmodulin through Coarse-Grained Simulations

Although the two domains of Calmodulin (CaM) are topologically similar, they differ in structural flexibility, stability, and Calcium binding affinity. Our coarse-grained simulations of the open-closed conformational transitions in Calcium-free Calmodulin (CaM) suggest kinetic scenarios in which the transition mechanisms for the two domains are distinct as well. Both domains have the same qualitative thermodynamic mechanisms: a “low temperature” two-state mechanism in which the domain remains relatively structured throughout the transition, and a “high temperature” mechanism involving a partially folded, unstable intermediate in the transition landscape. Under the same simulation conditions, the N-terminal domain (nCaM) exhibits two-state behavior, while the C-terminal domain (cCaM) populates the partially folded intermediate. The simulated transition rate for cCaM is much smaller due to the transient unfolding and refolding along the transition route compared to the two-state transition rate for nCaM. Differences in transition rates for the Calcium-free open/closed transition could explain measured binding rates of Ca2+ for the different domains. For example, slower conformational kinetics caused by the partially folded intermediate may bias cCaM towards a conformational selection binding mechanism, while nCaM maintains induced fit binding under some range of calcium concentration. Here, we explore such binding scenarios qualitatively.

Specific 12CβD212CγD2S13CεHD2 Isotopomer Labeling of Methionine To Characterize Protein Dynamics by 1H and 13C NMR Relaxation Dispersion

Protein dynamics on the micro- to millisecond time scale is increasingly found to be critical for biological function, as demonstrated by numerous NMR relaxation dispersion studies. Methyl groups are excellent probes of protein interactions and dynamics because of their favorable NMR relaxation properties, which lead to sharp signals in the 1H and 13C NMR spectra. Out of the six different methyl-bearing amino acid residue types in proteins, methionine plays a special role because of its extensive side-chain flexibility and the high polarizability of the sulfur atom. Methionine is over-represented in many protein–protein recognition sites, making the methyl group of this residue type an important probe of the relationships among dynamics, interactions, and biological function. Here we present a straightforward method to label methionine residues with specific 13CHD2 methyl isotopomers against a deuterated background. The resulting protein samples yield NMR spectra with improved sensitivity due to the essentially 100% population of the desired 13CHD2 methyl isotopomer, which is ideal for 1H and 13C spin relaxation experiments to investigate protein dynamics in general and conformational exchange in particular. We demonstrate the approach by measuring 1H and 13C CPMG relaxation dispersion for the nine methionines in calcium-free calmodulin (apo-CaM). The results show that the C-terminal domain, but not the N-terminal domain, of apo-CaM undergoes fast exchange between the ground state and a high-energy state. Since target proteins are known to bind specifically to the C-terminal domain of apo-CaM, we speculate that the high-energy state might be involved in target binding through conformational selection.

Rotational Dynamics of Calcium-Free Calmodulin Studied by 15N-NMR Relaxation Measurements

The backbone motions of calcium-free Xenopus calmodulin have been characterized by measurements of the 15N longitudinal relaxation times (T1) at 51 and 61 MHz, and by conducting transverse relaxation (T2), spin-locked transverse relaxation (T1ρ), and 15N-{1H} heteronuclear NOE measurements at 61 MHz 15N frequency. Although backbone amide hydrogen exchange experiments indicate that the N-terminal domain is more stable than calmodulin's C-terminal half, slowly exchanging backbone amide protons are found in all eight α-helices and in three of the four short β-strands. This confirms that the calcium-free form consists of stable secondary structure and does not adopt a ‘molten globule’ type of structure. However, the C-terminal domain of calmodulin is subject to conformational exchange on a time scale of about 350 μs, which affects many of the C-terminal domain residues. This results in significant shortening of the 15N T2 values relative to T1ρ, whereas the T1ρ and T2 values are of similar magnitude in the N-terminal half of the protein. A model in which the motion of the protein is assumed to be isotropic suggests a rotational correlation time for the protein of about 8 ns but quantitatively does not agree with the magnetic field dependence of the T1 values and does not explain the different T2 values found for different α-helices in the N-terminal domain. These latter parameters are compatible with a flexible dumbbell model in which each of calmodulin's two domains freely diffuse in a cone with a semi-angle of about 30° and a time constant of about 3 ns, whereas the overall rotation of the protein occurs on a much slower time scale of about 12 ns. The difference in the transverse relaxation rates observed between the amides in helices C and D suggests that the change in interhelical angle upon calcium binding is less than predicted by Herzberg et al. Strynadka and James [Strynadka, N. C. J. & James, M. N. G. (1988) Proteins Struct. Funct. Genet. 3, 1–17].

Monitoring Conformational Changes in Protein Complexes Using Chemical Cross-Linking and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: The Effect of Calcium Binding on the Calmodulin—Melittin Complex

Calmodulin is an EF hand calcium binding protein. Its binding affinities to various protein/peptide targets often depend on the conformational changes induced by the binding of calcium. One such target is melittin, which binds tightly to calmodulin in the presence of calcium, and inhibits its function. Chemical cross-linking combined with Fourier transform ion cyclotron resonance mass spectrometry has been employed to investigate the coordination of calmodulin and melittin in the complex at different concentrations of calcium. This methodology can be used to monitor structural changes of proteins induced by ligand binding, and study the effects these changes have on non- covalent interactions between proteins. Cross-linking results indicate that the binding place of the first melittin in the calcium free calmodulin form is the same as in the calcium loaded calmodulin/melittin complex.

Crystal structure of apo-calmodulin bound to the first two IQ motifs of myosin V reveals essential recognition features.

A 2.5-A resolution structure of calcium-free calmodulin (CaM) bound to the first two IQ motifs of the murine myosin V heavy chain reveals an unusual CaM conformation. The C-terminal lobe of each CaM adopts a semi-open conformation that grips the first part of the IQ motif (IQxxxR), whereas the N-terminal lobe adopts a closed conformation that interacts more weakly with the second part of the motif (GxxxR). Variable residues in the IQ motif play a critical role in determining the precise structure of the bound CaM, such that even the consensus residues of different motifs show unique interactions with CaM. This complex serves as a model for the lever arm region of many classes of unconventional myosins, as well as other IQ motif-containing proteins such as neuromodulin and IQGAPs.

Apo calmodulin binding to the L-type voltage-gated calcium channel Ca v1.2 IQ peptide

The influx of calcium through the L-type voltage-gated calcium channels (LTCCs) is the trigger for the process of calcium-induced calcium release (CICR) from the sarcoplasmic recticulum, an essential step for cardiac contraction. There are two feedback mechanisms that regulate LTCC activity: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF), both of which are mediated by calmodulin (CaM) binding. The IQ domain (aa 1645–1668) housed within the cytoplasmic domain of the LTCC Ca v1.2 subunit has been shown to bind both calcium-loaded (Ca 2+CaM ) and calcium-free CaM (apoCaM). Here, we provide new data for the structural basis for the interaction of apoCaM with the IQ peptide using NMR, revealing that the apoCaM C-lobe residues are most significantly perturbed upon complex formation. In addition, we have employed transmission electron microscopy of purified LTCC complexes which shows that both apoCaM and Ca 2+CaM can bind to the intact channel.

Who's Got the Calcium?

The human cardiac voltage-gated sodium channel (hH1) is essential for normal heart function, and mutations in the alpha subunit of the channel cause deadly arrhythmias. Some of these mutations are in a region that confers critical regulation of channel function by calcium, but exactly how calcium regulates the channel has been unclear. The hH1 protein contains an EF-hand calcium-binding motif that may bind calcium directly and an IQ-type calmodulin-binding motif that could confer calcium-dependent changes through changes in calmodulin. Shah et al. explored interactions between these domains to help clarify the roles of these direct and indirect responses to calcium in channel function. The authors characterized the binding properties of various channel domains and mutants by monitoring 15N-3H heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra. The EF-hand domain alone appeared to have a very low affinity for calcium (0.6 mM) that would be unlikely to have a physiological role. However, interaction of the IQ motif with the EF-hand domain enhanced calcium-binding affinity into the micromolar range. Contrary to expectations, a mutation in the EF-hand domain that causes arrhythmias in humans didn't alter calcium affinity but rather reduced affinity of the channel for binding calmodulin. On the other hand, a mutation in the IQ calmodulin-binding motif had little effect on affinity for calmodulin but reduced calcium-binding affinity. Also unusual was the finding that calcium-loaded calmodulin actually had lower affinity for an IQ motif-containing peptide than did calcium-free calmodulin. The authors propose that the effects of calcium can be explained as follows: In basal conditions, calmodulin is bound to the channel through the IQ motif. Increased concentrations of intracellular calcium weaken this interaction such that calmodulin is released, enabling the IQ motif to interact with the EF-hand domain, thus favoring calcium-dependent modulation of the channel through direct calcium binding. Further understanding of the intricate nature of these calcium-modulated domain interactions may reveal new therapeutic strategies to manage cardiac arrhythmias and other diseases associated with altered function of other members of this channel family in other cell types. V. N. Shah, T. L. Wingo, K. L. Weiss, C. K. Williams, J. R. Balser, W. J. Chazin, Calcium-dependent regulation of the voltage-gated sodium channel hH1: Intrinsic and extrinsic sensors use a common molecular switch. Proc. Natl. Acad. Sci. U.S.A. 103 , 3592-3597 (2006). [Abstract] [Full Text]

Interaction between calcium-free calmodulin and IQ motif of neurogranin studied by nuclear magnetic resonance spectroscopy

The interaction of Ca 2+-free calmodulin (apoCaM) with the IQ motif corresponding to the calmodulin-binding domain of neurogranin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of uncomplexed apoCaM and apoCaM in complex with the IQ motif recorded at 750 MHz were studied and the backbone assignments of the protein in both forms were obtained by triple-resonance multidimensional NMR experiments. Chemical shift perturbations were used to map the binding surfaces. Only a single set of resonances was observed throughout the titration, indicating that the binding interaction is under fast exchange. Analysis of chemical shift changes indicates that (a) the main interaction and conformational changes occur in the C-terminal domain of calmodulin and (b) linker-1 (residues 40–44) between EF-1 and EF-2, linker-3 (residues 112–117) between EF-3 and EF-4, and the end of the α-helix H (residues 145–148) may be involved in the binding process. The dissociation constant ( K d), estimated by fitting the chemical shift changes against the IQ peptide concentration, ranged from about 1.2×10 −5 to 8.8×10 −5 M. This result demonstrates that the interaction falls into the weak binding regime.

Molecular Dynamics Simulations of Calcium-Free Calmodulin in Solution

A 4-ns molecular dynamics simulation of calcium-free calmodulin in solution has been performed, using Ewald summation to treat electrostatic interactions. Our simulation results were mostly consistent with solution experimental studies, including NMR, fluorescence and x-ray scattering. The secondary structures within the N- and C-terminal domains were conserved in the simulation, with trajectory structures similar to the NMR-derived model structure 1CFD. However, the relative orientations of the domains, for which there are no NMR restraints, differed in details between the simulation and the 1CFD model. The most interesting information provided by the simulations is that the dynamics of calcium-free calmod- ulin in solution is dominated by slow rigid body reorientations of the domains. The interdomain distance fluctuated between 29 and 39 Å, and interdomain orientation angle, defined as the pseudo-dihedral formed by the four calcium binding sites, varied between −2° and 108°. Similarly, the domain linker region also exhibited significant fluctuations, with its length varying in the 34–45 Å range and its bend angle in the 10–100° range. The simulations are in accord with fluorescence results suggesting that calcium-free calmodulin is more compact and more flexible than the calcium activated form. Surprisingly, quite similar solvent accessibilities of the hydrophobic patches were seen in the calcium-free trajectory described in this work and previously generated calcium-loaded calmodulin simulations. Thus, our simulations suggest a reexamination of the standard model of the structural change of calmodulin upon calcium binding, involving exposure of the hydrophobic patches to solvent.

Why Calmodulin Has Two Lobes

The calcium-binding protein calmodulin (CaM) is thought to modulate the P/Q-type calcium channel of neurons in two ways: to facilitate and then to inhibit channel opening. DeMaria et al . have fleshed out the molecular details of this dual regulation and report that the two competing processes are accommodated by calcium binding to two different lobes of CaM. Calcium binding to the COOH-terminal lobe stimulated channel opening, whereas calcium binding to the NH 2 -terminal lobe inhibited channel activity in transfected cells. They also found that calcium-bound CaM exerted these effects through a region called IQ in the receptor's alpha subunit, and not a previously identified region called the CaM binding site. The authors further discovered that calcium-free CaM appears preassociated with the resting channel and this may facilitate a rapid response to calcium. C. D. DeMaria, T. W. Soong, B. A. Alseikhan, R. S. Alvania, D. T. Yue, Calmodulin bifurcates the local Ca 2+ signal that modulates P/Q-type Ca 2+ channels. Nature 411 : 484-489 (2001). [Online Journal]

Calcium Binding to Calmodulin Leads to an N-terminal Shift in Its Binding Site on the Ryanodine Receptor

The skeletal muscle calcium release channel, ryanodine receptor, is activated by calcium-free calmodulin and inhibited by calcium-bound calmodulin. Previous biochemical studies from our laboratory have shown that calcium-free calmodulin and calcium bound calmodulin protect sites at amino acids 3630 and 3637 from trypsin cleavage (Moore, C. P., Rodney, G., Zhang, J. Z., Santacruz-Toloza, L., Strasburg, G., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). We now demonstrate that both calcium-free calmodulin and calcium-bound calmodulin bind with nanomolar affinity to a synthetic peptide matching amino acids 3614-3643 of the ryanodine receptor. Deletion of the last nine amino acids (3635-3643) destroys the ability of the peptide to bind calcium-free calmodulin, but not calcium-bound calmodulin. We propose a novel mechanism for calmodulin's interaction with a target protein. Our data suggest that the binding sites for calcium-free calmodulin and calcium-bound calmodulin are overlapping and, when calcium binds to calmodulin, the calmodulin molecule shifts to a more N-terminal location on the ryanodine receptor converting it from an activator to an inhibitor of the channel. This region of the ryanodine receptor has previously been identified as a site of intersubunit contact, suggesting the possibility that calmodulin regulates ryanodine receptor activity by regulating subunit-subunit interactions.

Apocalmodulin Binds to the Myosin Light Chain Kinase Calmodulin Target Site

The interaction of a 20-residue-long peptide derived from the calmodulin-binding domain of the smooth muscle myosin light chain kinase with calcium-free calmodulin (apocalmodulin) was studied using a combination of isothermal titration calorimetry and differential scanning calorimetry. We showed that: (i) a significant binding between apocalmodulin and the target peptide (RS20) exists in the absence of salt (Ka = 10(6) M-1), (ii) the peptide interacts with the C-terminal lobe of calmodulin and adopts a partly helical conformation, and (iii) the presence of salt weakens the affinity of the peptide for apocalmodulin, emphasizing the importance of electrostatic interactions in the complex. Based on these results and taking into account the work of Bayley et al. (Bayley, P. M., Findlay, W.A., and Martin, S. R. (1996) Protein Sci. 5, 1215-1228), we suggest a physiological role for apocalmodulin.

Spin-locked multiple quantum coherence for signal enhancement in heteronuclear multidimensional NMR experiments

For methine sites the relaxation rate of 13C-1H two-spin coherence is generally slower than the relaxation rate of the individual 13C and 1H single spin coherences. The slower decay of two-spin coherence can be used to increase the sensitivity and resolution in heteronuclear experiments, particularly those that require correlation of H alpha and C alpha chemical shifts. To avoid dephasing of the two-spin coherence caused by 1H-1H J-couplings, the 1H spin is locked by the application of a weak rf field, resulting in a spin-locked multiple quantum coherence. For a sample of calcium-free calmodulin, use of the multiple quantum approach yields significant signal enhancement over the conventional constant-time 2D HSQC experiment. The approach is applicable to many multidimensional NMR experiments, as demonstrated for a 3D 13C-separated ROESY CT-HMQC spectrum.

Rotational dynamics of calcium-free calmodulin studied by 15N-NMR relaxation measurements.

The backbone motions of calcium-free Xenopus calmodulin have been characterized by measurements of the 15N longitudinal relaxation times (T1) at 51 and 61 MHz, and by conducting transverse relaxation (T2), spin-locked transverse relaxation (T1 rho), and 15N-[1H] heteronuclear NOE measurements at 61 MHz 15N frequency. Although backbone amide hydrogen exchange experiments indicate that the N-terminal domain is more stable than calmodulin's C-terminal half, slowly exchanging backbone amide protons are found in all eight alpha-helices and in three of the four short beta-strands. This confirms that the calcium-free form consists of stable secondary structure and does not adopt a 'molten globule' type of structure. However, the C-terminal domain of calmodulin is subject to conformational exchange on a time scale of about 350 microseconds, which affects many of the C-terminal domain residues. This results in significant shortening of the 15N T2 values relative to T1 rho, whereas the T1 rho and T2 values are of similar magnitude in the N-terminal half of the protein. A model in which the motion of the protein is assumed to be isotropic suggests a rotational correlation time for the protein of about 8 ns but quantitatively does not agree with the magnetic field dependence of the T1 values and does not explain the different T2 values found for different alpha-helices in the N-terminal domain. These latter parameters are compatible with a flexible dumb-bell model in which each of calmodulin's two domains freely diffuse in a cone with a semi-angle of about 30 degrees and a time constant of about 3 ns, whereas the overall rotation of the protein occurs on a much slower time scale of about 12 ns. The difference in the transverse relaxation rates observed between the amides in helices C and D suggests that the change in interhelical angle upon calcium binding is less than predicted by Herzberg et al. Strynadka and James [Strynadka, N. C. J. & James, M. N. G. (1988) Proteins Struct. Funct. Genet. 3, 1-17].

Measurement of HN-H? J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods

Two new methods are described for the measurement of three-bond JHNH alpha couplings in proteins isotopically enriched with 15N. Both methods leave the water magnetization in an unsaturated state, parallel to the z-axis, and therefore offer significant enhancements in sensitivity for rapidly exchanging backbone amide protons. The J couplings can be measured either from a set of constant-time 2D 1H-15N HMQC spectra, which are modulated in intensity by JHNH alpha, or from a water-flip-back version of the 3D HNHA experiment. The method is demonstrated for a sample of calcium-free calmodulin. Residues Lys75-Asp80 have JHNH alpha values in the 6-7 Hz range, suggesting that a break in the 'central helix' occurs at the same position as previously observed in solution NMR studies of Ca(2+)-ligated calmodulin.

Crystal growth of plant calmodulin

Large amounts of maize calmodulin have been isolated, sufficient for crystal growth under different conditions. No crystals have ever been obtained of calcium-free calmodulin. Polyethyleneglycol 6000 as the precipitant lead to crystals of variable form. In the presence of 50%–60% (v/v) 2-methyl-2,4-pentanediol, needle-like or plate-like crystals were obtained up to 150 μm in length; the average isodiametric crystal size was 30×25×15 μm 3, prior to seeding. During the growth phase, the plate-like calmodulin crystals show faces (flat, stepped and kinked), similar to that as predicted by the periodic bond chain theory.

Cleavage of phosphorylase kinase and calcium-free calmodulin by HIV-1 protease

Phosphorylase kinase and calcium-free calmodulin are digested by human immunodeficiency virus-1 protease. In phosphorylase kinase, the α subunit is preferentially hydrolyzed at arg 748-val 749. The β subunit is cleaved only slowly at leu 678-pro 679, and calmodulin, the integral δ subunit of phosphorylase kinase, is not cleaved at all. However, free calmodulin in the calcium-depleted form showed to be a good substrate for the protease. Here the cleavage occurs at phe 65-pro 66 and met 71-met 72. This fast hydrolysis of free calmodulin can be blocked by micromolar concentrations of Ca 2+ or millimolar concentrations of Mg 2+.

Calcium‐free calmodulin is a substrate of proteases from human immunodeficiency viruses 1 and 2

Calcium-free calmodulin-(CaM) is rapidly hydrolyzed by proteases from both human immunodeficiency viruses (HIV) 1 and 2. Kinetic analysis reveals a sequential order of cleavage by both proteases which initiates in regions of the molecule known from X-ray crystallographic analysis of Ca2+/CaM to be associated with calcium binding. Although HIV-1 and HIV-2 proteases hydrolyze two bonds in common, the initial site of cleavage required for subsequent events differs in each case. The first bond hydrolyzed by the HIV-1 protease is the Asn-Tyr linkage in the sequence, -N-I-D-G-D-G-Q-V-N-Y-E-E-, found in the fourth calcium binding loop. In contrast, it is an Ala-Ala bond in the third calcium loop, -D-K-D-G-N-G-Y-I-S-A-A-E-, that is first hydrolyzed by the HIV-2 enzyme, followed in short order by cleavage of the same Asn-Tyr linkage described above. Thereafter, both enzymes proceed to hydrolyze additional peptide bonds, some in common, some not. Considerable evidence exists that inhibitors are bound to the protease in an extended conformation and yet all of the cleavages we observed occur within, or at the beginning of helices in Ca2+/CaM, regions that also appear to be insufficiently exposed for protease binding. Molecular modeling studies indicate that CaM in solution must adopt a conformation in which the first cleavage site observed for each enzyme is unshielded and extended, and that subsequent cleavages involve further unwinding of helices.(ABSTRACT TRUNCATED AT 250 WORDS)