Calcium-loaded Calmodulin Research Articles

Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites

The autoinhibited plasma membrane calcium ATPase ACA8 from A. thaliana has an N-terminal autoinhibitory domain. Binding of calcium-loaded calmodulin at two sites located at residues 42–62 and 74–96 relieves autoinhibition of ACA8 activity.Through activity studies and a yeast complementation assay we investigated wild-type (WT) and N-terminally truncated ACA8 constructs (Δ20, Δ30, Δ35, Δ37, Δ40, Δ74 and Δ100) to explore the role of conserved motifs in the N-terminal segment preceding the calmodulin binding sites. Furthermore, we purified WT, Δ20- and Δ100-ACA8, tested activity in vitro and performed structural studies of purified Δ20-ACA8 stabilized in a lipid nanodisc to explore the mechanism of autoinhibition.We show that an N-terminal segment between residues 20 and 35 including conserved Phe32, upstream of the calmodulin binding sites, is important for autoinhibition and the activation by calmodulin. Cryo-EM structure determination at 3.3 Å resolution of a beryllium fluoride inhibited E2 form, and at low resolution for an E1 state combined with AlphaFold prediction provide a model for autoinhibition, consistent with the mutational studies.

Quantitative Agreement between Conformational Substates of Holo Calcium-Loaded Calmodulin Detected by Double Electron–Electron Resonance EPR and Predicted by Molecular Dynamics Simulations

Calcium-loaded calmodulin (CaM/4Ca2+) comprises two domains that undergo rigid body reorientation from a predominantly extended conformation to a compact one upon binding target peptides. A recent replica-exchange molecular dynamics (MD) simulation on holo CaM/4Ca2+ suggested the existence of distinct structural clusters (substates) along the path from extended to compact conformers in the absence of substrates. Here, we experimentally demonstrate the existence of CaM/4Ca2+ substates trapped in local minima by three freezing/annealing regimes (slow, 40 s; intermediate, 1.5 s; fast, 0.5 ms) using pulsed Q-band double electron-electron resonance (DEER) EPR spectroscopy to measure interdomain distances between nitroxide spin-labels positioned at A17C and A128C in the N- and C-terminal domains, respectively. The DEER echo curves were directly fit to population-optimized P(r) pairwise distance distributions calculated from the coordinates of the MD clusters and compact crystal structure. DEER data on fully deuterated CaM/4Ca2+ were acquired at multiple values of the second echo period (10-35 μs) and analyzed globally to eliminate instrumental and overfitting artifacts and ensure accurate populations, peak positions, and widths. The DEER data for all three freezing regimes are quantitatively accounted for within experimental error by 5-6 distinct conformers comprising a predominantly populated extended form (60-75%) and progressively more compact states whose populations decrease as the degree of compactness increases. The shortest interdomain separation is found in the compact crystal structure, which has an occupancy of 4-6%. Thus, CaM/4Ca2+ samples high energy local minima comprising a few discrete substates of increasing compactness in a rugged energy landscape.

Response of Terahertz Protein Vibrations to Ligand Binding: Calmodulin-Peptide Complexes as a Case Study.

Terahertz vibrations are sensitive reporters of the structure and interactions of proteins. Ligand binding alters the nature and distribution of these collective vibrations. The ligand-induced changes in the terahertz protein vibrations contribute to the binding entropy and to the overall thermodynamic stability of the resultant protein-ligand complexes. Here, we have examined the response of the low-frequency (below 6 terahertz) collective vibrations of the calcium-loaded calmodulin (CaM) to binding to five different ligands, both in the presence and absence of water, using normal-mode analysis and molecular dynamics simulations. A comparison of the vibrational spectra of hydrated and dry systems reveals that protein-solvent interactions stiffen the terahertz protein vibrations and that these solvent-coupled collective vibrations contribute significantly to the hydration-sensitive variation in the vibrational entropy of CaM. In the absence of water, the low-frequency vibrations of CaM are stiffened by ligand binding. On the contrary, the number and the cumulative vibrational entropy of low-frequency vibrational modes (ω < 200 cm-1) of the hydrated CaM are increased noticeably after binding to the peptides, indicating binding-induced softening of collective vibrations of the protein. Although the calculated and experimental binding affinities of the chosen complexes correlated reasonably well, no systematic correlation was observed between the protein vibrational entropy and the binding affinity. The results underscored the importance of the interplay of protein-ligand and solvent interactions in modulating the low-frequency vibrations of proteins.

High-affinity tamoxifen analogues retain extensive positional disorder when bound to calmodulin.

Using a combination of NMR and fluorescence measurements, we have investigated the structure and dynamics of the complexes formed between calcium-loaded calmodulin (CaM) and the potent breast cancer inhibitor idoxifene, a derivative of tamoxifen. High-affinity binding ( nM) saturates with a complex. The complex is an ensemble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches of CaM but, in contrast with the lower-affinity antagonists TFP, J-8, and W-7, does not substantially occupy the hydrophobic pocket. At least four idoxifene orientations per domain of CaM are necessary to satisfy the intermolecular nuclear Overhauser effect (NOE) restraints, and this requires that the idoxifene molecules switch rapidly between positions. The CaM molecule is predominantly in the form where the N and C-terminal domains are in close proximity, allowing for the idoxifene molecules to contact both domains simultaneously. Hence, the complex illustrates how high-affinity binding occurs without the loss of extensive positional dynamics.

Crystal Structures of Calcium-Loaded Calmodulin in Complex with C-Terminal Domains of Voltage-Gated Sodium Channels

Calmodulin (CaM) is a ubiquitous calcium sensor that can influence the gating of several ion channels. In both voltage-gated sodium (NaV) and calcium channels (CaV), CaM can affect channel inactivation. The primary binding site is located in the C-terminal region (CT), which consists of an IQ domain downstream of an EF-hand domain. We solved a crystal structure of a fully Ca2+-occupied CaM, bound to the CT of NaV1.5. We find the C-terminal lobe to bind to a site ∼90° rotated relative to a previous site that has been reported for apoCaM in complex with the NaV1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs changes the orientation of the EF-hand domain relative to the IQ domain, such that the EF-hand domain would clash with a Ca2+-occupied C-lobe of CaM. This highlights the role of the EF-hand domain in modulating the binding mode of CaM. The related NaV1.4 channel has been shown to undergo Ca2+-dependent inactivation (CDI) akin to CaVs. We were unable to crystallize the NaV1.4 CT with Ca2+/CaM, but instead solved a crystal structure of Ca2+/CaM bound to the NaV1.4 IQ domain. Superposing this with NaV CT structures shows that Ca2+/CaM would clash with the EF-hand domain, suggesting that binding of Ca2+/CaM to the NaV1.4 CT would cause a reorientation of the EF-hand domain. We postulate that this event is a possible conformational switch that underlies CDI.

Rotamer Jumps, Proton Exchange, and Amine Inversion Dynamics of Dimethylated Lysine Residues in Proteins Resolved by pH-Dependent 1H and 13C NMR Relaxation Dispersion.

Post-translational methylation of lysine side chains is of great importance for protein regulation, including epigenetic control. Here, we present specific 13CHD2 labeling of dimethylated lysines as a sensitive probe of the structure, interactions, and dynamics of these groups, and outline a theoretical and experimental framework for analyzing their conformational dynamics using 1H and 13C CPMG relaxation dispersion experiments. Dimethylated lysine side chains in calcium-loaded calmodulin show a marked pH dependence of their Carr-Purcell-Meiboom-Gill (CPMG) dispersion profiles, indicating complex exchange behavior. Combined analysis of 1H and 13C CPMG relaxation dispersions requires consideration of 12-state correlated exchange of the two methyl groups due to circular three-state rotamer jumps around the Cε-Nζ axis combined with proton exchange and amine inversion. Taking into account a number of fundamental constraints, the exchange model can be reduced to include only three fitted parameters, namely, the geometric average of the rotamer-jump rate constants, the rate constant of deprotonation of Nζ, and the chemical shift difference between the trans and gauge positions of the 13C or 1H nuclei. The pH dependence indicates that protonation of the end group dramatically slows down rotamer exchange for some lysine residues, whereas deprotonation leads to rapid amine inversion coupled with rotamer scrambling. The observed variation among residues in their exchange behavior appears to depend on the structural environment of the side chain. Understanding this type of exchange process is critical to correctly interpreting NMR spectra of methylated lysine side chains. The exchange model presented here forms the basis for studying the structure and dynamics of epigenetically modified lysine side chains and perturbations caused by changes in pH or interactions with target proteins.

1H, 15N, and 13C chemical shift assignments of the regulatory domain of human calcineurin

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM-CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete 13C, 15N and 1H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of 13C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A 15N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.

Generating a Model for Calmodulin Induced Folding in the Disordered Regulatory Domain of Calcineurin

Calcineurin (CaN) plays an important role in the T-cell activation, cardiac system development and nervous system function. Previous studies have suggested that the regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the regulatory domain, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is at least partially disordered, and binding of CaM induces folding in the RD. Previous studies have shown that an α-helical structure forms in the N-terminal half of the RD, but organization may occur in the C-terminal half as well. Here, we are interested in the structural transition of the full length RD as it binds to CaM. Using nuclear magnetic resonance (NMR) spectroscopy, we have successfully assigned >85% of the 15N, 13Cα, 13Cβ and HN chemical shifts of the unbound, regulatory domain of CaN. While the protein is disordered, secondary chemical shifts indicate that some regions possess α-helical propensity, even in the unbound state. Our study of the CaM and CaN interaction supports the formation of a distal helix in the region between the AID and calmodulin-binding region. Heat capacity changes upon binding predict that 54 residues fold when CaM binds to CaN, consistent with the formation of this distal helix. Paramagnetic relaxation enhancement (PRE) studies of this interaction suggest a potential binding mode where the distal helix binds to CaM near residues I9-A15. Mutagenesis in the distal helix disrupts PREs, further supporting this hypothesis. Together, these data suggest that the interactions between CaM and the distal helix of CaN can be important in regulation of phosphatase activity.

NMR Investigation of Calmodulin Induced Folding in the Regulatory Domain of Calcineurin

Calcineurin (CaN) plays an important role in the T-cell activation, cardiac system development and nervous system function. Previous studies have suggested that the 97-residue regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end. Calcium/Calmodulin activates the serine/threonine phosphatase activity of CaN by binding to the regulatory domain, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto inhibitory domain from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is at least partially disordered, and binding of CaM induces folding in the RD. Previous studies have shown that an α-helical structure forms in the N-terminal half of the RD, but organization may occur in the C-terminal half as well. Here we are interested in the structural transition of the full length RD as it binds to CaM. Using nuclear magnetic resonance (NMR) spectroscopy, we have successfully assigned >85% of the 15N, 13Cα, 13Cβ and HN chemical shifts of the unbound, full-length regulatory domain of CaN. While the protein is unstructured, secondary chemical shifts indicate some regions with α-helical propensity, even in the free state. At present, we are studying how the spectrum changes as calcium-loaded CaM is added to the solution. In the long term, this work will identify how binding-induced folding changes the RD structure, which could ultimately lead to a better understanding of how CaM activates CaN.

Stoichiometry of the calcineurin regulatory domain-calmodulin complex.

Calcineurin is an essential serine/threonine phosphatase that plays vital roles in neuronal development and function, heart growth, and immune system activation. Calcineurin is unique in that it is the only phosphatase known to be activated by calmodulin in response to increasing intracellular calcium concentrations. Calcium-loaded calmodulin binds to the regulatory domain of calcineurin, resulting in a conformational change that removes an autoinhibitory domain from the active site of the phosphatase. We have determined a 1.95 Å crystal structure of calmodulin bound to a peptide corresponding to its binding region from calcineurin. In contrast to previous structures of this complex, our structure has a stoichiometry of 1:1 and has the canonical collapsed, wraparound conformation observed for many calmodulin-substrate complexes. In addition, we have used size-exclusion chromatography and time-resolved fluorescence to probe the stoichiometry of binding of calmodulin to a construct corresponding to almost the entire regulatory domain from calcineurin, again finding a 1:1 complex. Taken in sum, our data strongly suggest that a single calmodulin protein is necessary and sufficient to bind to and activate each calcineurin enzyme.

Pulsed EPR Distance Measurements Resolve the Impact of Site-Specific Calmodulin Methionine Oxidation

We have examined the structural impact of oxidizing specific protein methionines (Met) in the C-ter lobe of calmodulin (CaM); these oxidation sites are known to abolish CaM regulation of the major calcium release channel, the ryanodine receptor complex (RyR). Protein oxidation by reactive oxygen species (ROS) is strongly associated with loss of strength in skeletal muscle and is proposed to play major roles in aging and degenerative muscle disease.We have linked oxidation-induced changes in RyR regulation to changes in CaM-RyR structure using (1) protein mutagenesis to mimic oxidation at specific sites and (2) spectroscopy to resolve oxidation-induced changes in protein structural dynamics. Pulsed EPR distance measurements across CaM's lobes (multiple pairs of labeling sites, one label on each lobe) were sensitive to large-scale conformational changes that accompany both calcium binding and RyR peptide binding. In the absence of calcium, CaM was highly disordered, populating a broad distribution of conformations. Calcium binding strongly stabilized the elongated conformation, while RyR peptide binding to calcium-loaded CaM strongly stabilized the compact conformation. CaM conformation, particularly the distribution over structural states, was sensitive to Met to Gln substitutions (M109Q and M124Q) designed to mimic CaM Met oxidation. Structural sensitivity to M-to-Q mutations was observed in both the presence and absence of calcium, and in complex with RyR peptide. We conclude that Met oxidation alters CaM's functional interaction with RyR through changes in the orientation and flexibility of CaM's lobes.This work is supported by a University of Wisconsin-La Crosse Faculty Research Grant to JC Klein, NIH grants to DD Thomas (R37AG026160 and P30AR057220), RJ Moen (F31AG037303), the Minnesota Biophysical Spectroscopy Center and the Minnesota Supercomputing Institute.

Designing Molecular Dynamics Simulations to Shift Populations of the Conformational States of Calmodulin

We elucidate the mechanisms that lead to population shifts in the conformational states of calcium-loaded calmodulin (Ca2+-CaM). We design extensive molecular dynamics simulations to classify the effects that are responsible for adopting occupied conformations available in the ensemble of NMR structures. Electrostatic interactions amongst the different regions of the protein and with its vicinal water are herein mediated by lowering the ionic strength or the pH. Amino acid E31, which is one of the few charged residues whose ionization state is highly sensitive to pH differences in the physiological range, proves to be distinctive in its control of population shifts. E31A mutation at low ionic strength results in a distinct change from an extended to a compact Ca2+-CaM conformation within tens of nanoseconds, that otherwise occur on the time scales of microseconds. The kinked linker found in this particular compact form is observed in many of the target-bound forms of Ca2+-CaM, increasing the binding affinity. This mutation is unique in controlling C-lobe dynamics by affecting the fluctuations between the EF-hand motif helices. We also monitor the effect of the ionic strength on the conformational multiplicity of Ca2+-CaM. By lowering the ionic strength, the tendency of nonspecific anions in water to accumulate near the protein surface increases, especially in the vicinity of the linker. The change in the distribution of ions in the vicinal layer of water allows N- and C- lobes to span a wide variety of relative orientations that are otherwise not observed at physiological ionic strength. E31 protonation restores the conformations associated with physiological environmental conditions even at low ionic strength.

Calmodulin Adopts an Extended Conformation when Interacting with L-Selectin in Membranes

Calmodulin, an intracellular calcium-binding protein, is thought to regulate ectodomain shedding of many membrane proteins, but the underlying molecular mechanism has remained unclear. Basing on a solution structure of calcium-loaded calmodulin in complex with a L-selectin fragment that contains a portion of its transmembrane domain, Gifford et al. (University of Calgary) recently suggested that calmodulin regulates L-selectin shedding by binding directly to a portion of the L-selectin transmembrane domain in a compact conformation. Using fluorescently labeled calmodulin, we show however that calmodulin adopts a distinctly different and much more extended conformation when it binds to the CLS peptide (i.e. the entire transmembrane and cytoplasmic domains of L-selectin) reconstituted in the phosphatidylcholine liposome with micromolar dissociation constant and in a calcium-independent manner. Calmodulin adopts a similarly extended conformation in a ternary complex with the N-terminal FERM domain of moesin and CLS reconstituted in the phospholipid liposome that mimics the native membrane environment. These results indicate that calmodulin does not bind directly to the transmembrane domain of L-selectin. Understanding the association of calmodulin with L-selectin helps to shed light on the mechanisms underlying regulation of ectodomain shedding.

Role of Primary Structure Variation in Structure and Function of Calcineurin

A. fumigatus is one of the leading causes of invasive fungal infections in solid organ transplant and adult stem cell transplant recipients. It is estimated that over 20% of Americans are infected with T. gondii. T. gondii is life threatening in immunocompromised patients. Here, we study the enzyme calcineurin that is critical for the virulence and pathogenicity of these organisms. CaN is activated when calcium‐loaded calmodulin (CaM) binds the regulatory domain (RD) of the A subunit. Upon binding to CaM, the RD of human calcineurin transitions from a disordered state to an ordered state, gaining alpha helical structure. Research done in our lab has determined that the C‐terminus of the RD forms a helix (distal helix) that is necessary for the normal function of human CaN. In A. fumigatus and T. gondii, the sequence for this helix is deleted. A. fumigatus and T. gondii CaN contain modular insertions and deletions in the RD. A Ser/Pro rich region (SPRR) found in the RD that is unique to A. fumigatus is critical for the function of CaN and survival. The phosphorylation of this SPRR has also been shown to be critical in the pathogenicity of the organism. Investigations into the protein sequence of CaN of T. gondii show a unique insertion in the same area as the SPRR in A. fumigatus, but the function of this insertion is unknown. Thus, we are probing into how structure affects function through evolutionary variations.

Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase, SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation.

A Calmodulin Interacting Protein Iqcg Is Required for Definitive Hematopoiesis in Zebrafish

AbstractAbstract 764We previously identified a novel fusion gene NUP98-IQCG in a bi-phenotypic acute T-lymphoid/myeloid leukemia patient. However, the function of IQCG has never been demonstrated. By taking advantage of the zebrafish as a model organism, we investigated the role of IQCG in hematopoietic development and found that the definitive hematopoiesis was severely impaired in the iqcg deficient embryos. The hematopoietic stem cell (HSC) population as well as the number of multilineage differentiated hematopoietic cells was reduced. Moreover, the number of phosphorelated histone3 positive HSC was decreased, which indicated a compromised proliferation in iqcg deficient HSC.Since IQCG protein contains an IQ calmodulin binding motif (IQ motif), we next examined if IQCG protein can interact with calmodulin. Co-immunoprecipitation assay results showed that both human and zebrafish IQCG proteins have the ability to precipitate calmodulin. Deletion of IQ motif disrupted this interaction. The isothermal titration calorimetry results indicated that the IQ motif of IQCG protein have a lower association constant with calcium-loaded calmodulin than apo-calmodulin. We next crystallized the complex of calmodulin and IQ motif of IQCG in two circumstances-with and without calcium. Determination of these two crystal structures indicated different interaction details between them. These results suggest that IQCG may be involved in calcium signaling in HSC.Previous studies reported that calmodulin dependent protein kinase IV (CaMKIV) involves in HSC maintenance and survival. So we wondered if CaMKIV acts downstream of IQCG. Indeed, whole-mount immunofluorescence data showed that the level of phosphorelated CaMKIV markedly reduced in iqcg morphants compared with controls. Reactivation of CaMKIV by co-injection with constitutively activated CaMKIV (CACaMKIV) mRNA rescued the HSC number in iqcg deficient embryos. Furthermore, embryos injected with camkIV morpholino phenocopied the iqcg morphants.In summary, we demonstrate an important role of the novel NUP98 partner gene IQCG in definitive hematopoietic development. IQCG may control the HSC proliferation through interacting with calmodulin and acting upstream of CaMKIV, which suggests a novel pathway in definitive hematopoietic development. Disclosures:No relevant conflicts of interest to declare.

A bimodular mechanism of calcium control in eukaryotes

Calcium ions (Ca(2+)) have an important role as secondary messengers in numerous signal transduction processes, and cells invest much energy in controlling and maintaining a steep gradient between intracellular (∼0.1-micromolar) and extracellular (∼2-millimolar) Ca(2+) concentrations. Calmodulin-stimulated calcium pumps, which include the plasma-membrane Ca(2+)-ATPases (PMCAs), are key regulators of intracellular Ca(2+) in eukaryotes. They contain a unique amino- or carboxy-terminal regulatory domain responsible for autoinhibition, and binding of calcium-loaded calmodulin to this domain releases autoinhibition and activates the pump. However, the structural basis for the activation mechanism is unknown and a key remaining question is how calmodulin-mediated PMCA regulation can cover both basal Ca(2+) levels in the nanomolar range as well as micromolar-range Ca(2+) transients generated by cell stimulation. Here we present an integrated study combining the determination of the high-resolution crystal structure of a PMCA regulatory-domain/calmodulin complex with in vivo characterization and biochemical, biophysical and bioinformatics data that provide mechanistic insights into a two-step PMCA activation mechanism mediated by calcium-loaded calmodulin. The structure shows the entire PMCA regulatory domain and reveals an unexpected 2:1 stoichiometry with two calcium-loaded calmodulin molecules binding to different sites on a long helix. A multifaceted characterization of the role of both sites leads to a general structural model for calmodulin-mediated regulation of PMCAs that allows stringent, highly responsive control of intracellular calcium in eukaryotes, making it possible to maintain a stable, basal level at a threshold Ca(2+) concentration, where steep activation occurs.

Transient, Sparsely Populated Compact States of Apo and Calcium-Loaded Calmodulin Probed by Paramagnetic Relaxation Enhancement: Interplay of Conformational Selection and Induced Fit

Calmodulin (CaM) is the universal calcium sensor in eukaryotes, regulating the function of numerous proteins. Crystallography and NMR show that free CaM-4Ca(2+) exists in an extended conformation with significant interdomain separation, but clamps down upon target peptides to form a highly compact structure. NMR has revealed substantial interdomain motions in CaM-4Ca(2+), enabled by a flexible linker. In one instance, CaM-4Ca(2+) has been crystallized in a compact configuration; however, no direct evidence for transient interdomain contacts has been observed in solution, and little is known about how large-scale interdomain motions contribute to biological function. Here, we use paramagnetic relaxation enhancement (PRE) to characterize transient compact states of free CaM that are too sparsely populated to observe by traditional NMR methods. We show that unbound CaM samples a range of compact structures, populated at 5-10%, and that Ca(2+) dramatically alters the distribution of these configurations in favor of states resembling the peptide-bound structure. In the absence of Ca(2+), the target peptide binds only to the C-terminal domain, and the distribution of compact states is similar with and without peptide. These data suggest an alternative pathway of CaM action in which CaM remains associated with its kinase targets even in the resting state. Only CaM-4Ca(2+), however, shows an innate propensity to form the physiologically active compact structures, suggesting that Ca(2+) activates CaM not only through local structural changes within each domain but also through more global remodeling of interdomain interactions. Thus, these findings illustrate the subtle interplay between conformational selection and induced fit.

Subtle pH differences trigger single residue motions for moderating conformations of calmodulin

This study reveals the essence of ligand recognition mechanisms by which calmodulin (CaM) controls a variety of Ca(2+) signaling processes. We study eight forms of calcium-loaded CaM each with distinct conformational states. Reducing the structure to two degrees of freedom conveniently describes main features of the conformational changes of CaM via simultaneous twist-bend motions of the two lobes. We utilize perturbation-response scanning (PRS) technique, coupled with molecular dynamics simulations. PRS is based on linear response theory, comprising sequential application of directed forces on selected residues followed by recording the resulting protein coordinates. We analyze directional preferences of the perturbations and resulting conformational changes. Manipulation of a single residue reproduces the structural change more effectively than that of single/pairs/triplets of collective modes of motion. Our findings also give information on how the flexible linker acts as a transducer of binding information to distant parts of the protein. Furthermore, by perturbing residue E31 located in one of the EF hand motifs in a specific direction, it is possible to induce conformational change relevant to five target structures. Independently, using four different pK(a) calculation strategies, we find this particular residue to be the charged residue (out of a total of 52), whose ionization state is most sensitive to subtle pH variations in the physiological range. It is plausible that at relatively low pH, CaM structure is less flexible. By gaining charged states at specific sites at a pH value around 7, such as E31 found in the present study, local conformational changes in the protein will lead to shifts in the energy landscape, paving the way to other conformational states. These findings are in accordance with Fluorescence Resonance Energy Transfer (FRET) measured shifts in conformational distributions towards more compact forms with decreased pH. They also corroborate mutational studies and proteolysis results which point to the significant role of E31 in CaM dynamics.

A 1.3-Å Structure of Zinc-bound N-terminal Domain of Calmodulin Elucidates Potential Early Ion-binding Step

Calmodulin (CaM) is a 16.8-kDa calcium-binding protein involved in calcium-signal transduction. It is the canonical member of the EF-hand family of proteins, which are characterized by a helix–loop–helix calcium-binding motif. CaM is composed of N- and C-terminal globular domains (N-CaM and C-CaM), and within each domain there are two EF-hand motifs. Upon binding calcium, CaM undergoes a significant, global conformational change involving reorientation of the four helix bundles in each of its two domains. This conformational change upon ion binding is a key component of the signal transduction and regulatory roles of CaM, yet the precise nature of this transition is still unclear. Here, we present a 1.3-Å structure of zinc-bound N-terminal calmodulin (N-CaM) solved by single-wavelength anomalous diffraction phasing of a selenomethionyl N-CaM. Our zinc-bound N-CaM structure differs from previously reported CaM structures and resembles calcium-free apo-calmodulin (apo-CaM), despite the zinc binding to both EF-hand motifs. Structural comparison with calcium-free apo-CaM, calcium-loaded CaM, and a cross-linked calcium-loaded CaM suggests that our zinc-bound N-CaM reveals an intermediate step in the initiation of metal ion binding at the first EF-hand motif. Our data also suggest that metal ion coordination by two key residues in the first metal-binding site represents an initial step in the conformational transition induced by metal binding. This is followed by reordering of the N-terminal region of the helix exiting from this first binding loop. This conformational switch should be incorporated into models of either stepwise conformational transition or flexible, dynamic energetic state sampling-based transition.