Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin

We have used circular dichroism and frequency-domain fluorescence spectroscopy to determine how the site-specific substitution of Tyr138 with either Phe138 or Gln138 affects the structural coupling between the opposing domains of calmodulin (CaM). A double mutant was constructed involving conservative substitution of Tyr99 --> Trp99 and Leu69 --> Cys69 to assess the structural coupling between the opposing domains, as previously described [Sun, H., Yin, D., and Squier, T. C. (1999) Biochemistry 38, 12266-12279]. Trp99 acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements to probe the conformation of the central helix. Cys69 provides a reactive group for the covalent attachment of 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), which functions as a FRET acceptor and permits the measurement of the rotational dynamics of the amino-terminal domain. These CaM mutants demonstrate normal calcium-dependent gel-mobility shifts and changes in their near-UV CD spectra, have similar secondary structures to wild-type CaM following calcium activation, and retain the ability to fully activate the plasma membrane Ca-ATPase. The global folds, therefore, of both the carboxyl- and amino-terminal domains in these CaM mutants are similar to that of wild-type CaM. However, in comparison to wild-type CaM, the substitution of Tyr138 with either Phe138 or Gln138 results in (i) alterations in the average spatial separation and increases in the conformational heterogeneity between the opposing globular domains and (ii) the independent rotational dynamics of the amino-terminal domain. These results indicate that alterations in either the hydrogen bond between Tyr138 and Glu82 or contact interactions between aromatic amino acid side chains have the potential to initiate the structural collapse of CaM normally associated with target protein binding and activation.     

Closer proximity between opposing domains of vertebrate calmodulin following deletion of Met(145)-Lys(148)

To investigate the structural linkage between the opposing globular domains in vertebrate calmodulin (CaM), we have constructed a CaM mutant (CaMX(145)) deficient in the last four amino acids between Met(145) and Lys(148) at the carboxyl terminal. Circular dichroism and fluorescence spectroscopic measurements were used to detect changes in the average secondary and tertiary structure of CaMX(145) in comparison to full-length CaM. Complementary measurements of the maximal calcium-binding stoichiometry and ability to activate the plasma membrane (PM) Ca-ATPase permit an assessment of the functional significance of observed structural changes. In comparison with native CaM, we find that CaMX(145) exhibits (i) a large reduction in alpha-helical content, (ii) a dramatic decrease in the average spatial separation between the opposing globular domains, (iii) the loss of one high-affinity calcium-binding site, and (iv) a diminished binding affinity for the PM-Ca-ATPase. Thus, the sequence near the carboxyl terminus functions to stabilize high-affinity calcium binding at one site and facilitates important intramolecular interactions that maintain CaM in an extended conformation. However, despite the large conformational changes resulting from deletion of the last four amino acids at the carboxyl terminal, CaMX(145) can fully activate the PM-Ca-ATPase. These results indicate that target protein binding can restore the nativelike structure critical to function, emphasizing that the structure of the central helix is not critical to CaM function under equilibrium conditions. Rather, the central helix functions to maintain the spatial separation between the opposing domains in CaM that may be critical to high-affinity binding and the rapid activation of the PM-Ca-ATPase, which are necessary for optimal calcium signaling. Thus, following initial association between CaM and target proteins, structural changes involving the carboxyl-terminal sequence have the potential to play an important role in triggering the structural collapse of CaM that facilitates the rapid and cooperative binding of the opposing globular domains with target proteins, which is important to high-affinity binding and rapid enzyme activation.     

Dynamic motion of helix A in the amino-terminal domain of calmodulin is stabilized upon calcium activation

Calcium-dependent changes in the internal dynamics and average structures of the opposing globular domains of calmodulin (CaM), as well as their relative spatial arrangement, contribute to the productive association between CaM and a range of different target proteins, affecting their functional activation. To identify dynamic structural changes involving individual alpha-helical elements and their modulation by calcium activation, we have used site-directed mutagenesis to engineer a tetracysteine binding motif within helix A near the amino terminus of calmodulin (CaM), permitting the selective and rigid attachment of the fluorescent probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) with full retention of function. The rigid tetracoordinate linkage of FlAsH to CaM, in conjunction with frequency domain fluorescence anisotropy measurements, allows assessment of dynamic changes associated with calcium activation without interference from independent probe motion. Taking advantage of the large fluorescence enhancement associated with binding of FlAsH to CaM, we determined rates of binding of FlAsH to apo-CaM and calcium-activated CaM to be 2800 +/- 80 and 310 +/- 10 M(-)(1) s(-)(1), respectively. There is no difference in the solvent accessibility of the bound FlAsH irrespective of calcium binding to CaM. Thus, given that FlAsH selectively labels disordered structures, the large difference in rates of FlAsH binding indicates that calcium binding stabilizes helix A. Frequency domain anisotropy measurements of bound FlAsH indicate that prior to calcium activation, helix A undergoes large amplitude nanosecond motions. Following calcium activation, helix A becomes immobile, and structurally coupled to the overall rotation of CaM. We discuss these results in the context of a model that suggests stabilization of helix A relative to other domain elements in the CaM structure is critical to defining high-affinity binding clefts, and in promoting specific and ordered binding of the opposing lobes of CaM to target proteins.     

Structural uncoupling between opposing domains of oxidized calmodulin underlies the enhanced binding affinity and inhibition of the plasma membrane Ca-ATPase

Stabilization of the plasma membrane Ca-ATPase (PMCA) in an inactive conformation upon oxidation of multiple methionines in the calcium regulatory protein calmodulin (CaM) is part of an adaptive cellular response to minimize ATP utilization and the generation of reactive oxygen species (ROS) under conditions of oxidative stress. To differentiate oxidant-induced structural changes that selectively modify the amino-terminal domain of CaM from those that modulate the conformational coupling between the opposing domains, we have engineered a tetracysteine binding motif within helix A in the amino-terminal domain of calmodulin (CaM) that permits the selective and rigid attachment of the conformationally sensitive fluorescent probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)(2) (FlAsH-EDT(2)). The position of the FlAsH label in the amino-terminal domain provides a signal for monitoring its binding to the CaM-binding sequence of the PMCA. Following methionine oxidation, there is an enhanced binding affinity between the amino-terminal domain and the CaM-binding sequence of the PMCA. To identify oxidant-induced structural changes, we used frequency domain fluorescence anisotropy measurements to assess the structural coupling between helix A and the amino- and carboxyl-terminal domains of CaM. Helix A undergoes large amplitude motions in apo-CaM; following calcium activation, helix A is immobilized as part of a conformational switch that couples the opposing domains of CaM to stabilize the high-affinity binding cleft associated with target protein binding. Methionine oxidation disrupts the structural coupling between opposing globular domains of CaM, without affecting the calcium-dependent immobilization of helix A associated with activation of the amino-terminal domain to promote high-affinity binding to target proteins. We suggest that this selective disruption of the structural linkage between the opposing globular domains of CaM relieves steric constraints associated with high-affinity target binding, permitting the formation of new contact interactions between the amino-terminal domain and the CaM-binding sequence that stabilizes the PMCA in an inhibited conformation.     

A 1.3-A 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.     

Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4

Human apolipoprotein E (apoE) isoforms exhibit different conformational stabilities and lipid-binding properties that give rise to altered cholesterol metabolism among the isoforms. Using Trp-substituted mutations and site-directed fluorescence labeling, we made a comprehensive comparison of the conformational organization of the N- and C-terminal domains and lipid interactions between the apoE3 and apoE4 isoforms. Trp fluorescence measurements for selectively Trp-substituted variants of apoE isoforms demonstrated that apoE4 adopts less stable conformations in both the N- and C-terminal domains compared to apoE3. Consistent with this, the conformational reorganization of the N-terminal helix bundle occurs at lower guanidine hydrochloride concentration in apoE4 than in apoE3 as monitored by fluorescence resonance energy transfer (FRET) from Trp residues to acrylodan attached at the N-terminal helix. Upon binding of apoE3 and apoE4 variants to egg phosphatidylcholine small unilamellar vesicles, similar changes in Trp fluorescence or FRET efficiency were observed for the isoforms, indicating that the opening of the N-terminal helix bundle occurs similarly in apoE3 and apoE4. Introduction of mutations into the C-terminal domain of the apoE isoforms to prevent self-association and maintain the monomeric state resulted in great increase in the rate of binding of the C-terminal helices to a lipid surface. Overall, our results demonstrate that the different conformational organizations of the N- and C-terminal domains have a minor effect on the steady-state lipid-binding behavior of apoE3 and apoE4: rather, self-association property is a critical determinant in the kinetics of lipid binding through the C-terminal helices of apoE isoforms.     

Calcium-dependent stabilization of the central sequence between Met(76) and Ser(81) in vertebrate calmodulin

Spin-label electron paramagnetic resonance (EPR) provides optimal resolution of dynamic and conformational heterogeneity on the nanosecond time-scale and was used to assess the structure of the sequence between Met(76) and Ser(81) in vertebrate calmodulin (CaM). Previous fluorescence resonance energy transfer and anisotropy measurements indicate that the opposing domains of CaM are structurally coupled and the interconnecting central sequence adopts conformationally distinct structures in the apo-form and following calcium activation. In contrast, NMR data suggest that the opposing domains of CaM undergo independent rotational dynamics and that the sequence between Met(76) and Ser(81) in the central sequence functions as a flexible linker that connects two structurally independent domains. However, these latter measurements also resolve weak internuclear interactions that suggest the formation of transient helical structures that are stable on the nanosecond time-scale within the sequence between Met(76) and Asp(80) in apo-CaM (H. Kuboniwa, N. Tjandra, S. Grzekiek, H. Ren, C. B. Klee, and A. Bax, 1995, Nat. Struct. Biol. 2:768-776). This reported conformational heterogeneity was resolved using site-directed mutagenesis and spin-label EPR, which detects two component spectra for 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate spin labels (MTSSL) bound to CaM mutants T79C and S81C that include a motionally restricted component. In comparison to MTSSL bound within stable helical regions, the fractional contribution of the immobilized component at these positions is enhanced upon the addition of small amounts of the helicogenic solvent trifluoroethanol (TFE). These results suggest that the immobilized component reflects the formation of stable secondary structures. Similar spectral changes are observed upon calcium activation, suggesting a calcium-dependent stabilization of the secondary structure. No corresponding changes are observed in either the solvent accessibility to molecular oxygen or the maximal hyperfine splitting. In contrast, more complex spectral changes in the line-shape and maximal hyperfine splitting are observed for spin labels bound to sites that undergo tertiary contact interactions. These results suggest that spin labels at solvent-exposed positions within the central sequence are primarily sensitive to backbone fluctuations and that either TFE or calcium binding stabilizes the secondary structure of the sequence between Met(76) and Ser(81) and modulates the structural coupling between the opposing domains of CaM.     

Phosphorylation by cAMP-dependent protein kinase modulates the structural coupling between the transmembrane and cytosolic domains of phospholamban

We have used frequency-domain fluorescence spectroscopy to investigate the structural linkage between the transmembrane and cytosolic domains of the regulatory protein phospholamban (PLB). Using an engineered PLB having a single cysteine (Cys(24)) derivatized with the fluorophore N-(1-pyrenyl)maleimide (PMal), we have used fluorescence resonance energy transfer (FRET) to measure the average spatial separation and conformational heterogeneity between PMal bound to Cys(24) in the transmembrane domain and Tyr(6) in the cytosolic domain near the amino terminus of PLB. In these measurements, PMal serves as a FRET donor, and Tyr(6) serves as a FRET acceptor following its nitration by tetranitromethane. The native structure of PLB is retained following site-directed mutagenesis and chemical modification, as indicated by the ability of the derivatized PLB to fully regulate the Ca-ATPase following their co-reconstitution. To assess how phosphorylation modulates the structure of PLB itself, FRET measurements were made following reconstitution of PLB in membrane vesicles made from extracted sarcoplasmic reticulum membrane lipids. We find that the cytosolic domain of PLB assumes a wide range of conformations relative to the transmembrane sequence, consistent with other structural data indicating the presence of a flexible hinge region between the transmembrane and cytosolic domains of PLB. Phosphorylation of Ser(16) by PKA results in a 3 A decrease in the spatial separation between PMal at Cys(24) and nitroTyr(6) and an almost 2-fold decrease in conformational heterogeneity, suggesting a stabilization of the hinge region of PLB possibly through an electrostatic linkage between phosphoSer(16) and Arg(13) that promotes a coil-to-helix transition. This structural transition has the potential to function as a conformational switch, since inhibition of the Ca-ATPase requires disruption of the secondary structure of PLB in the vicinity of the hinge element to permit association with the nucleotide binding domain at a site located approximately 50 A above the membrane surface. Following phosphorylation, the stabilization of the helical content in the hinge domain will disrupt this inhibitory interaction by reducing the maximal dimension of the cytosolic domain of PLB. Thus, stabilization of the structure of PLB following phosphorylation of Ser(16) is part of a switching mechanism, which functions to alter binding interactions between PLB and the nucleotide binding domain of the Ca-ATPase that modulates enzyme inhibition.     

Direct sugar binding to LacY measured by resonance energy transfer

Trp151 in the lactose permease of Escherichia coli (LacY) is an important component of the sugar-binding site and the only Trp residue out of six that is in close proximity to the galactopyranoside in the structure (1PV7). The short distance between Trp151 and the sugar is favorable for F?rster resonance energy transfer (FRET) to nitrophenyl or dansyl derivatives with the fluorophore at the anomeric position of galactose. Modeling of 4-nitrophenyl-alpha-d-galactopyranoside (alpha-NPG) in the binding-site of LacY places the nitrophenyl moiety about 12 A away from Trp151, a distance commensurate with the F?rster distance for a Trp-nitrobenzoyl pair. We demonstrate here that alpha-NPG binding to LacY containing all six native Trp residues causes galactopyranoside-specific FRET from Trp151. Moreover, binding of alpha-NPG is sufficiently slow to resolve time-dependent fluorescence changes by stopped-flow. The rate of change in Trp --> alpha-NPG FRET is linearly dependent upon sugar concentration, which allows estimation of kinetic parameters for binding. Furthermore, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) covalently attached to the cytoplasmic end of helix X is sensitive to sugar binding, reflecting a ligand-induced conformational change. Stopped-flow kinetics of Trp --> alpha-NPG FRET and sugar-induced changes in MIANS fluorescence in the same protein reveal a two-step process: a relatively rapid binding step detected by Trp151 --> alpha-NPG FRET followed by a slower conformational change detected by a change in MIANS fluorescence.     

Oxidatively modified calmodulin binds to the plasma membrane Ca-ATPase in a nonproductive and conformationally disordered complex

Oxidation of either Met(145) or Met(146) in wheat germ calmodulin (CaM) to methionine sulfoxide prevents the CaM-dependent activation of the plasma membrane (PM) Ca-ATPase (D. Yin, K. Kuczera, and T. C. Squier, 2000, Chem. Res. Toxicol. 13:103-110). To investigate the structural basis for the inhibition of the PM-Ca-ATPase by oxidized CaM (CaM(ox)), we have used circular dichroism (CD) and fluorescence spectroscopy to resolve conformational differences within the complex between CaM and the PM-Ca-ATPase. The similar excited-state lifetime and solvent accessibility of the fluorophore N-1-pyrenyl-maleimide covalently bound to Cys(26) in unoxidized CaM and CaM(ox) indicates that the globular domains within CaM(ox) assume a native-like structure following association with the PM-Ca-ATPase. However, in comparison with oxidized CaM there are increases in the 1) molar ellipticity in the CD spectrum and 2) conformational heterogeneity between the opposing globular domains for CaM(ox) bound to the CaM-binding sequence of the PM-Ca-ATPase. Furthermore, CaM(ox) binds to the PM-Ca-ATPase with high affinity at a distinct, but overlapping, site to that normally occupied by unoxidized CaM. These results suggest that alterations in binding interactions between CaM(ox) and the PM-Ca-ATPase block important structural transitions within the CaM-binding sequence of the PM-Ca-ATPase that are normally associated with enzyme activation.     

Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli.

cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached to Cys178. Binding of cAMP in the CRP-(cAMP)2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP-(cAMP)4 complex alters the surroundings of Trp85. Time-resolved anisotropy measurements indicated that cAMP binding in the CRP-(cAMP)2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP-(cAMP)2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 A in apo-CRP to 18.7 A in the CRP-(cAMP)2 complex. The binding of cAMP in the CRP-(cAMP)4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP-DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to approximately 20 A. The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein.     

Helix A stabilization precedes amino-terminal lobe activation upon calcium binding to calmodulin

The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)resorufin (ReAsH), which upon binding to an engineered tetracysteine motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the (1)H- (15)N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe ( K d = 0.36 +/- 0.04 microM), which results in a reduction in the rate of ReAsH binding from 4900 M (-1) s (-1) to 370 M (-1) s (-1). In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe require calcium occupancy of amino-terminal sites (K d = 18 +/- 3 microM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain linker that release helix A, thereby facilitating the formation of calcium binding sites in the amino-terminal lobe and linked tertiary structural rearrangements to form a high-affinity binding cleft that can associate with target proteins.     

A mutation in the lactose permease of Escherichia coli that decreases conformational flexibility and increases protein stability

Lactose permease with Cys154 --> Gly (helix V) binds substrate with high affinity but catalyzes little or no transport. The purified, detergent-solubilized mutant protein exhibits much greater thermal stability than the wild type and little tendency to aggregate. Stabilization is also observed in vivo with an unstable mutant that is expressed at significantly higher levels when the Cys154 --> Gly mutation is introduced. In addition, ligand-induced conformational changes are markedly reduced or abolished by the Cys154 --> Gly mutation: (i) Although the fluorescence of purified single Trp33 (helix I) permease is enhanced by ligand binding, introduction of the Cys154 --> Gly mutation abolishes the effect. (ii) The rate of 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) labeling of permease with a single Cys residue in place of Val331 (helix X) is increased in the presence of ligand but reduced when the Cys154 --> Gly mutation is present. (iii) Fluorescence emission intensity of MIANS-labeled single Cys331 permease is enhanced and blue shifted in the Cys154 --> Gly mutant background, indicating that the latter mutation causes position 331 to become exposed to a less polar environment. The results indicate that the Cys154 --> Gly mutation causes a more compact structure and decreased conformational flexibility, an alteration that specifically blocks the structural changes necessary for substrate translocation with little or no effect on ligand binding.     

Calmodulin’s flexibility allows for promiscuity in its interactions with target proteins and peptides

The small bilobal calcium regulatory protein calmodulin (CaM) activates numerous target enzymes in response to transient changes in intracellular calcium concentrations. Binding of calcium to the two helix-loop-helix calcium-binding motifs in each of the globular domains induces conformational changes that expose a methionine-rich hydrophobic patch on the surface of each domain of the protein, which it uses to bind to peptide sequences in its target enzymes. Although these CaM-binding domains typically have little sequence identity, the positions of several bulky hydrophobic residues are often conserved, allowing for classification of CaM-binding domains into recognition motifs, such as the 1–14 and 1–10 motifs. For calcium-independent binding of CaM, a third motif known as the IQ motif is also common. Many CaM-peptide complexes have globular conformations, where CaM’s central linker connecting the two domains unwinds, allowing the protein to wrap around a single predominantly α-helical target peptide sequence. However, novel structures have recently been reported where the conformation of CaM is highly dissimilar to these globular complexes, in some instances with less than a full compliment of bound calcium ions, as well as novel stoichiometries. Furthermore, many divergent CaM isoforms from yeast and plant species have been discovered with unique calcium-binding and enzymatic activation characteristics compared to the single CaM isoform found in mammals.     

Functional significance of the central helix in calmodulin

The 3-A crystal structure of calmodulin indicates that it has a polarized tertiary arrangement in which calcium binding domains I and II are separated from domains III and IV by a long central helix consisting of residues 65-92. To investigate the functional significance of the central helix, mutated calmodulins were engineered with alterations in this region. Using oligonucleotide-primed site-directed mutagenesis, Thr-79 was converted to Pro-79 to generate CaMPM. CaMPM was further mutated by insertion of Pro-Ser-Thr-Asp between Asp-78 and Pro-79 to yield CaMIM. Calmodulin, CaMPM, and CaMIM were indistinguishable in their ability to activate calcineurin and Ca2+-ATPase. All mutated calmodulins would also maximally activate cGMP-phosphodiesterase and myosin light chain kinase, however, the concentrations of CaMPM and CaMIM necessary for half-maximal activation (Kact) were 2- and 9-fold greater, respectively, than CaM23. Conversion of the 2 Pro residues in CaMIM to amino acids that predict retention of helical secondary structure did not restore normal calmodulin activity. To investigate the nature of the interaction between mutated calmodulins and target enzymes, synthetic peptides modeled after the calmodulin binding region of smooth and skeletal muscle myosin light chain kinase were prepared and used as inhibitors of calmodulin-dependent cGMP-phosphodiesterase. The data suggest that the different kinetics of activation of myosin light chain kinase by CaM23 and CaMIM are not due to differences in the ability of the activators to bind to the calmodulin binding site of this enzyme. These observations are consistent with a model in which the length but not composition of the central helix is more important for the activation of certain enzymes. The data also support the hypothesis that calmodulin contains multiple sites for protein-protein interaction that are differentially recognized by its multiple 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 (Ca²⁺)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 (Ca²⁺)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. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Conformational states and fluctuations in endothelial nitric oxide synthase under calmodulin regulation

Mechanisms that regulate nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions and is activated by calmodulin (CaM) binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two NOS electron transfer domains in an FRET dye-labeled endothelial NOS reductase domain (eNOSr) and to understand how CaM affects the dynamics to regulate catalysis by shaping the spatial and temporal conformational behaviors of eNOSr. In addition, we developed and applied a new imaging approach capable of recording three-dimensional FRET efficiency versus time images to characterize the impact on dynamic conformal states of the eNOSr enzyme by the binding of CaM, which identifies clearly that CaM binding generates an extra new open state of eNOSr, resolving more detailed NOS conformational states and their fluctuation dynamics. We identified a new output state that has an extra open conformation that is only populated in the CaM-bound eNOSr. This may reveal the critical role of CaM in triggering NOS activity as it gives conformational flexibility for eNOSr to assume the electron transfer output FMN-heme state. Our results provide a dynamic link to recently reported EM static structure analyses and demonstrate a capable approach in probing and simultaneously analyzing all of the conformational states, their fluctuations, and the fluctuation dynamics for understanding the mechanism of NOS electron transfer, involving electron transfer among FAD, FMN, and heme domains, during nitric oxide synthesis.     

Analysis of the functional coupling between calmodulin's calcium binding and peptide recognition properties

The enhancement of calmodulin's (CaM) calcium binding activity by an enzyme or a recognition site peptide and its diminution by key point mutations at the protein recognition interface (e.g., E84K-CaM), which is more than 20 A away from the nearest calcium ligation structure, can be described by an expanded version of the Adair-Klotz equation for multiligand binding. The expanded equation can accurately describe the calcium binding events and their variable linkage to protein recognition events can be extended to other CaM-regulated enzymes and can potentially be applied to a diverse array of ligand binding systems with allosteric regulation of ligand binding, whether by other ligands or protein interaction. The 1.9 A resolution X-ray crystallographic structure of the complex between E84K-CaM and RS20 peptide, the CaM recognition site peptide from vertebrate smooth muscle and nonmuscle forms of myosin light chain kinase, provides insight into the structural basis of the functional communication between CaM's calcium ligation structures and protein recognition surfaces. The structure reveals that the complex adapts to the effect of the functional mutation by discrete adjustments in the helix that contains E84. This helix is on the amino-terminal side of the helix-loop-helix structural motif that is the first to be occupied in CaM's calcium binding mechanism. The results reported here are consistent with a sequential and cooperative model of CaM's calcium binding activity in which the two globular and flexible central helix domains are functionally linked, and provide insight into how CaM's calcium binding activity and peptide recognition properties are functionally coupled.     

A Novel Trans Conformation of Ligand-Free Calmodulin

Calmodulin (CaM) is a highly conserved eukaryotic protein that binds specifically to more than 100 target proteins in response to calcium (Ca²⁺) signal. CaM adopts a considerable degree of structural plasticity to accomplish this physiological role; however, the nature and extent of this plasticity remain to be fully understood. Here, we report the crystal structure of a novel trans conformation of ligand-free CaM where the relative disposition of two lobes of CaM is different, a conformation to-date not reported. While no major structural changes were observed in the independent N- and C-lobes as compared with previously reported structures of Ca²⁺/CaM, the central helix was tilted by ∼90° at Arg75. This is the first crystal structure of CaM to show a drastic conformational change in the central helix, and reveals one of several possible conformations of CaM to engage with its binding partner.     

Activation of the edema factor of Bacillus anthracis by calmodulin: evidence of an interplay between the EF-calmodulin interaction and calcium binding

Calmodulin (CaM) is a remarkably flexible protein which can bind multiple targets in response to changes in intracellular calcium concentration. It contains four calcium-binding sites, arranged in two globular domains. The calcium affinity of CaM N-terminal domain (N-CaM) is dramatically reduced when the complex with the edema factor (EF) of Bacillus anthracis is formed. Here, an atomic explanation for this reduced affinity is proposed through molecular dynamics simulations and free energy perturbation calculations of the EF-CaM complex starting from different crystallographic models. The simulations show that electrostatic interactions between CaM and EF disfavor the opening of N-CaM domains usually induced by calcium binding. Relative calcium affinities of the N-CaM binding sites are probed by free energy perturbation, and dissociation probabilities are evaluated with locally enhanced sampling simulations. We show that EF impairs calcium binding on N-CaM through a direct conformational restraint on Site 1, by an indirect destabilization of Site 2, and by reducing the cooperativity between the two sites.