Crystal structure of calmodulin

The crystal structure of calmodulin has been determined to 3.6 A resolution. At this resolution the polypeptide chain can be traced. Some of the side chains have tentatively been identified. Refinement of the structure with x-ray diffraction data measured to 1.65 A resolution is continuing. As reported by Babu et al. calmodulin is about 65 A long and 30 A in diameter. Homolog domains 1 and 2 are related by a local twofold axis, as in parvalbumin and in troponin C, and form one end of the molecule. Domains 3 and 4 form the other end. The second alpha-helix of domain 2 and a short interdomain region are continuous with the first helix of domain 3, thereby forming a single helix from residues 67-93. The central region, residues 75-84, of this long helix forms a handle connecting the two pairs of homolog domains. Exclusive of the residues, 75-84, in the handle the closet approach of side chains of pair 1, 2 to pair 3, 4 is 12 A. The spatial relationship of pair 1, 2 to pair 3, 4 is similar in calmodulin to the relationship of the corresponding pairs in troponin C. However, in troponin C there are three additional residues in the handle region of the long alpha-helix and the two pairs are about 5.0 A further apart. On the surface of pair 1, 2 in calmodulin there is one extended region with many hydrophobic side chains from both domain 1 and domain 2. This hydrophobic patch is bounded by two distinct clusters of anionic side chains, one from the beginning of the first helix of domain 1 and on the other side of the hydrophobic surface one from the beginning of the first helix of domain 2. Homologously, the hydrophobic patch on the surface of pair 3, 4 is bounded by two clusters of aspartate and glutamate residues. Either or both of these hydrophobic surfaces may be sites to which calmodulin target proteins bind.     

4Ca2+.troponin C forms dimers in solution at neutral pH that dissociate upon binding various peptides: small-angle X-ray scattering studies of peptide-induced structural changes.

Small-angle X-ray scattering data have been measured for rabbit skeletal muscle troponin C and its complexes with the venom peptides melittin and mastoparan as well as synthetic peptides based on regions of the troponin I sequence implicated in troponin C binding. At the neutral pH used in this study (pH 6.8), troponin C shows a tendency to form dimers in the presence of 4 mol equiv of Ca2+, but is monomeric in solution when 2 or less mol equiv of Ca2+ is present. The 4Ca2+.troponin C dimers dissociate upon binding melittin, mastoparan, and peptides based on residues 96-115, 1-30, and 1-40 in the troponin I sequence. This result suggests that the peptide-binding sites overlap with the regions of contact between troponin C molecules forming a dimer. Like the structurally homologous calcium-binding protein calmodulin, troponin C shows conformational flexibility upon binding different peptides. Upon binding melittin, troponin C contracts in a similar manner to calmodulin when it binds peptides known to form amphiphilic helices (e.g., melittin, mastoparan, or MLCK-I). In contrast, mastoparan binding to troponin C does not result in a contracted structure. The scattering data indicate troponin C also remains in an extended structure upon binding the inhibitory peptides having the same sequence as residues 96-115 in troponin I.     

Probable role of amphiphilicity in the binding of mastoparan to calmodulin

Two-dimensional helical wheel diagrams and calculations of mean hydrophobic moments show mastoparan, mastoparan X, and Polistes mastoparan to have all the properties expected for amphiphilic helices. Circular dichroic properties are consistent with a random form for these peptides in dilute aqueous solution, but greater than 50% helix is apparent when the peptides are dissolved in 70% trifluoroethanol/water mixtures (v/v) or when the peptides are bound to calmodulin. Changes in the fluorescence spectra, anisotropy, and accessibility of tryptophan whose indole side chain is on the apolar surface of the amphiphilic helix imply a significant role for the apolar surface in the binding of the mastoparans and another amphiphilic peptide, melittin, to calmodulin. These data provide a useful model for designing high-affinity synthetic peptide inhibitors of calmodulin.     

The structure of melittin in the form I crystals and its implication for melittin's lytic and surface activities.

Melittin from bee venom is water-soluble, yet integrates into membranes and lyses cells. Each melittin chain consists of 26 amino acid residues and in aqueous salt solutions it exists as a tetramer. We have determined the molecular structure of the tetramer in two crystal forms grown from concentrated salt solutions. In both crystal forms the melittin polypeptide is a bent alpha-helical rod, with the "inner" surface largely consisting of hydrophobic sidechains and the "outer" surface consisting of hydrophilic side chains. Thus, the helix is strongly amphiphilic. In the tetramer, four such helices contribute their hydrophobic side chains to the center of the molecule. The packing of melittin tetramers is also very similar in the two crystal forms: they are packed in planar layers with the outsides forming hydrophilic surfaces and the insides (the centers of melittin tetramers) forming a hydrophobic surface. We suggest that the surface activity of melittin can be rationalized in terms of these surfaces. The lytic activity of melittin can also be interpreted in terms of the molecular structure observed in the crystals: the hydrophobic inner surface of a melittin helix may integrate into the apolar region of a bilayer with the helix axis approximately parallel to the plane of the bilayer, and with the hydrophilic surface exposed to the aqueous phase. This integration would be expected to disrupt the bilayer because of melittin helix would penetrate only a short distance into it. Additionally, the integration of melittin from one side of a bilayer would produce a surface area difference across the bilayer, perhaps leading to lysis. In this view, melittin is distinct from membrane proteins that penetrate evenly into both leaflets of a bilayer or exactly halfway through a bilayer, and hence we refer to melittin as a surface-active protein.     

A model for human cardiac troponin C and for modulation of its Ca2+ affinity by drugs

Calcium sensitizers are drugs which increase force development in striated muscle by sensitizing myofilaments to Ca2+. This can happen by increasing Ca2+ affinity of the regulatory domain of Ca2+ binding protein troponin C. High resolution crystal structures of two calcium binding proteins, calmodulin (Babu et al.: J. Mol. Biol. 203:191-204, 1988) and skeletal troponin C (Satyshur et al.: J. Biol. Chem. 263:1628-1647, 1988; Herzber et al.: J. Mol. Biol. 203:761-779, 1988), have recently been published. This makes it possible to model in detail the calcium-sensitizing action of drugs on troponin C. In this study a model of human cardiac troponin C in three-calcium state has been constructed. When calcium is bound to calcium site II of cardiac troponin C an open conformation of the protein results, which has a hydrophobic pocket surrounded by a few polar side chains. Complexation of three drugs, trifluoperazine, bepridil, and pimobendan, to the hydrophobic pocket is studied using energy minimization techniques. Two different binding modes are found, which differ in the location of a strong electrostatic interaction. In analogy with the crystal structure of skeletal troponin C it is hypothezed that in cardiac troponin C an interaction occurs between Gln-50 and Asp-88, which has a long-range effect on calcium binding. The binding modes of drugs, where a strong interaction with Asp-88 exists, can effectively prevent the interaction between Asp-88 and Gln-50 in the protein, and are proposed to be responsible for the calcium-sensitizing properties of the studied drugs.     

Converting troponin C into calmodulin: effects of mutations in the central helix and of changes in temperature

Calmodulin (CaM) and troponin C (TnC) are the most similar members of EF-hand family and show few differences in the primary structure. Here, we use mutants of troponin that mimic calmodulin and changes in temperature to investigate the factors that determine their specificity as regulatory proteins. Using a double mutant of troponin that resembles calmodulin in lacking both the N-terminal helix and KGK(91-93) we observe a small difference from troponin in binding to the erythrocyte Ca(2+)-ATPase, and an improvement in enzyme activation. A triple mutant, where in addition, the residues 88-90 are replaced with the corresponding sequence from calmodulin is equivalent to calmodulin in maximal activation, and it restores protein ability to increase Ca(2+) affinity for the enzyme. However, this mutant also binds less tightly (1/100) than calmodulin. Remarkably, a decrease in temperature has a more marked effect in protein binding than either mutation, reducing the difference in affinities to 18-fold, but without any improvement in their ability to increase Ca(2+) affinity for the enzyme. Spectroscopic analysis of hydrophobic domain exposure in EF-hand proteins was carried out using 8-anilino-1-naphthalenesulfonic acid (ANS). The probe shows a much higher fluorescence when bound to the complex Ca(4)-calmodulin than to Ca(4)-troponin. Decreasing the temperature exposes additional hydrophobic regions of troponin. Changing the Mg(2+) concentration does not affect their bindings to the enzyme. It is suggested that the requirements for troponin to mimic calmodulin in binding to the target enzyme, and those for activating it, are met by different regions of the protein.     

Calmodulin binding to alpha 1-purothionin: solution binding and modeling of the complex.

CD and fluorescence spectroscopic measurements show that calmodulin (CaM) binds to purothionins (alpha 1-purothionin: alpha 1-PT; beta-purothionin: beta-PT) in 1:1 stoichiometry with an affinity similar to that exhibited with the tightest binding CaM-binding peptides. Using the available crystal structures of CaM and alpha 1-PT, a model has been built for the interaction of CaM and alpha 1-PT and subjected to potential energy minimization. In the model, there is a bend in the central helix of CaM similar to that suggested by Persechini and Kretsinger (J. Card. Pharm. 12:501-512, 1988). alpha 1-PT fits snugly into the cavity formed by the bent CaM molecule with each of its two helices making apolar interactions with each of the two hydrophobic clefts situated at the terminal domains of CaM. The complex is further stabilized by numerous polar and electrostatic interactions on the rims of the clefts. Our model is compared with two other similar models previously reported for the CaM complexes with other helical peptides and generalizations about the mode of CaM binding to target proteins are made, which have wide relevance to the function of CaM. By analogy, a similar model is predicted for a CaM-beta-PT complex.     

Quantification of protein-protein interactions with chemical cross-linking and mass spectrometry

Chemical cross-linking in combination with mass spectrometry has largely been used to study protein structures and protein-protein interactions. Typically, it is used in a qualitative manner to identify cross-linked sites and provide a low-resolution topological map of the interacting regions of proteins. Here, we investigate the capability of chemical cross-linking to quantify protein-protein interactions using a model system of calmodulin and substrates melittin and mastoparan. Calmodulin is a well-characterized protein which has many substrates. Melittin and mastoparan are two such substrates which bind to calmodulin in 1:1 ratios in the presence of calcium. Both the calmodulin-melittin and calmodulin-mastoparan complexes have had chemical cross-linking strategies successfully applied in the past to investigate topological properties. We utilized an excess of immobilized calmodulin on agarose beads and formed complexes with varying quantities of mastoparan and melittin. Then, we applied disuccinimidyl suberate (DSS) chemical cross-linker, digested and detected cross-links through an LC-MS analytical method. We identified five interpeptide cross-links for calmodulin-melittin and three interpeptide cross-links for calmodulin-mastoparan. Using cross-linking sites of calmodulin-mastoparan, we demonstrated that mastoparan also binds in two orientations to calmodulin. We quantitatively demonstrated that both melittin and mastoparan preferentially bind to calmodulin in a parallel fashion, which is opposite to the preferred binding mode of the majority of known calmodulin binding peptides. We also demonstrated that the relative abundances of cross-linked peptide products quantitatively reflected the abundances of the calmodulin peptide complexes formed.     

Identification of Helical Packing Motifs Common to Bacteriorhodopsin and G Protein-Coupled Receptors

A sequence analysis and comparison of transmembrane helices in bacteriorhodopsin (BR) and G protein-coupled receptors (GPCRs) is presented to identify potential regions of homology across protein families. The results show a common pattern of residues is conserved within the interhelical contact regions of BR that fit a knob-into-hole structural motif previously postulated for globular proteins and photosynthetic reaction centers. Based on an alignment of conserved prolines in transmembrane helices, it is inferred that analogous helix packing arrangements are possible in the rhodopsin-like GPCRs. Molecular models of GPCR helices V and VI indicate these interactions occur between aromatic and hydrophobic residues flanking the highly conserved prolines in these sequences. A similar packing arrangement is shown to occur in the X-ray structure of the melittin which also displays a unique pairing of proline-linked helices. The contact pattern identified is further applied to predict the packing of pairs of proline-containing helices in the pheromone-like and cAMP GPCRs. A potential role in stabilizing structure formation is also suggested for the contacts. The results and conclusions are supported by recent biophysical studies of zinc binding to kappa-opioid receptor mutants.     

Identification of Helical Packing Motifs Common to Bacteriorhodopsin and G Protein-Coupled Receptors

A sequence analysis and comparison of transmembrane helices in bacteriorhodopsin (BR) and G protein-coupled receptors (GPCRs) is presented to identify potential regions of homology across protein families. The results show a common pattern of residues is conserved within the interhelical contact regions of BR that fit a knob-into-hole structural motif previously postulated for globular proteins and photosynthetic reaction centers. Based on an alignment of conserved prolines in transmembrane helices, it is inferred that analogous helix packing arrangements are possible in the rhodopsin-like GPCRs. Molecular models of GPCR helices V and VI indicate these interactions occur between aromatic and hydrophobic residues flanking the highly conserved prolines in these sequences. A similar packing arrangement is shown to occur in the X-ray structure of the melittin which also displays a unique pairing of proline-linked helices. The contact pattern identified is further applied to predict the packing of pairs of proline-containing helices in the pheromone-like and cAMP GPCRs. A potential role in stabilizing structure formation is also suggested for the contacts. The results and conclusions are supported by recent biophysical studies of zinc binding to kappa-opioid receptor mutants.     

Disulfide bond assignment and identification of regions required for functional activity of oncostatin M

Oncostatin M is a polypeptide cytokine having unique structure and diverse biological activities, including the ability to inhibit growth of certain cultured tumor cells. Here we have determined the disulfide bonding pattern of recombinant oncostatin M and have used site-directed mutagenesis to identify regions of this molecule necessary for receptor binding and growth inhibitory activities. Two intramolecular disulfide bonds, C6-C127 and C49-C167, were identified in recombinant oncostatin M. Analysis of mutations at each of the five cysteines in oncostatin M indicated that mutants C49S and C167S were inactive (less than 1/10 wild type activity) in growth inhibitory assays and radioreceptor assays. Carboxyl-terminal deletion mutations terminating at S185 and beyond were active, but further shortening abolished activity in both assays. Two deletion mutants proximal to C49 (delta 22-36 and delta 44-47) and insertion mutant GAG77 also were inactive. One deletion mutant, delta 87-90, had significantly (approximately 3-fold) increased activities in both growth inhibitory assays and radioreceptor assays. A potential amphiphilic domain was identified beginning at C167 and extending toward the carboxyl terminus. Two mutants having altered hydrophobic residues within this domain (F176G and F184G) were inactive, suggesting that these residues are required for proper conformation of the receptor binding site. Taken together, these results indicate that biological activity of oncostatin M requires discontinuous regions of the molecule, including residues near the essential disulfide bond, C49-C167, and within a putative amphiphilic helix at the carboxyl terminus. Oncostatin M thus belongs to a growing family of cytokines whose interactions with their respective receptors are mediated in part by known or predicted carboxyl-terminal amphiphilic helices.     

Static and kinetic studies of calmodulin and melittin complex.

Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.     

Affinity-purified melittin antibody recognizes the calmodulin-binding domain on calmodulin target proteins

Melittin is a 26-amino acid amphipathic peptide which binds to calmodulin in a calcium-dependent manner. The utility of melittin as a peptide replica of the calmodulin-binding region of calmodulin acceptor proteins (CaMBPs) was investigated. Antibody against melittin was raised and purified by antigen affinity chromatography. Interaction of the antibody with CaMBPs was initially suggested by the ability of anti-melittin-Sepharose, but not nonimmune IgG-Sepharose, to bind calmodulin-dependent cyclic AMP phosphodiesterase. Direct interaction of melittin antibody with the calmodulin-binding domain of acceptor proteins was demonstrated by quantitative inhibition of calmodulin binding to the purified CaMBPs, myosin light chain kinase, and eel electric organ CaMBP55. These results indicate that melittin antibody identifies regions of structural similarity between calmodulin acceptor proteins, and this region includes a common calmodulin-binding domain.     

Cloning and expression pattern of EPAS1 in the chicken embryo. Colocalization with tyrosine hydroxylase

The thermodynamics of interaction of two model peptides melittin and mastoparan with bovine brain calmodulin (CAM) and a smaller CAM analogue, a calcium binding protein from Entamoeba histolytica (CaBP) in 10 mM MOPS buffer (pH 7.0) was examined using isothermal titration calorimetry (ITC). These data show that CAM binds to both the peptides and the enthalpy of binding is endothermic for melittin and exothermic for mastoparan at 25 degrees C. CaBP binds to the longer peptide melittin, but does not bind to mastoparan, the binding enthalpy being endothermic in nature. Concurrently, we also observe a larger increase in alpha-helicity upon the binding of melittin to CAM when compared to CaBP. The role of hydrophobic interactions in the binding process has also been examined using 8-anilino-1-naphthalene-sulphonic acid (ANS) binding monitored by ITC. These results have been employed to rationalize the energetic consequences of the binding reaction.     

Effects of calmodulin and related proteins on the hemolytic activity of melittin

The calcium-dependent binding of melittin by calmodulin effectively inhibits the hemolytic activity of melittin in suspensions of washed rabbit erythrocytes. Protection is also obtained with troponin C (+/-Ca++), denatured phosphorylase kinase, and denatured calcineurin but not with whole troponin or the native enzymes. These effects can be used both in assays for melittin in venom samples and in determinations of calmodulin or related proteins.     

Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin

CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C-terminal domain of calmodulin. Four discrete regions of CyaA bind calcium-loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an alpha-helix of CyaA makes extensive contacts with the calcium-induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin-induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA-calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.     

Ca-inhibited binding of melittin with parvalbumin. A new role for parvalbumins?

It was found that pike parvalbumins pI 4.2 and 5.0 bind amphiphilic peptide melittin extracted from bee venom in an extraordinary Ca-dependent manner: in apo-state the protein forms a tight equimolar complex with melittin (Ka = 10(6) M-1 at 18 degrees C); in Ca- (and Mg-) loaded state it does not take place. Heating of the protein up to temperatures above the denaturation temperature of apo-parvalbumin does not change the stoichiometry of the complex but increases its association constant by an order of magnitude (Ka = 1.2.10(7) M-1 at 44 degrees C). Isolated Ca-binding domain of parvalbumin, 38-108, retains the ability for Ca-inhibited binding of equimolar quantities of melittin. The possible function of parvalbumin in vivo is suggested: Ca-inhibited interactions with some intracellular components.     

Duchenne muscular dystrophy and dystrophin: Sequence homology observations

Duchenne muscular dystrophy (DMD) is a genetically transmitted disease characterized by progressive muscle weakness and usually leads to death. DMD results from the absence, deficiency or dysfunction of the protein dystrophin. Analysis of protein data bases, including homology alignments and domain recognition patterns, have located highly significant correlations between dystrophin and other calcium regulating proteins. In particular, a major portion of the dystrophin sequence has been found to contain repeating units of approximately 100 amino acid residues. These repeating units were found to exhibit significant homology to troponin I. Troponin I has been found to bind to the calcium binding proteins calmodulin and troponin C. The regions of highest homology were characterized by patterns of high localization of charged amino acids and thus could represent a possible calmodulin or troponin C surface accessible binding site. Since subcellular localization studies have indicated that dystrophin is associated with the triadic junction, these findings imply that dystrophin could be involved in controlling intracellular calcium homeostasis.Special issue dedicated to Dr. Lawrence Austin.     

Distribution of distances between the tryptophan and the N-terminal residue of melittin in its complex with calmodulin, troponin C, and phospholipids.

We used frequency-domain measurements of fluorescence resonance energy transfer to measure the distribution of distances between Trp-19 of melittin and a 1-dimethylamino-5-sulfonylnaphthalene (dansyl) residue on the N-terminal-alpha-amino group. Distance distributions were obtained for melittin free in solution and when complexed with calmodulin (CaM), troponin C (TnC), or palmitoyloleoyl-L-alpha-phosphatidylcholine (POPC) vesicles. A wide range of donor (Trp-19)-to-acceptor (dansyl) distances was found for free melittin, which is consistent with that expected for the random coil state, characterized by a Gaussian width (full width at half maxima) of 28.2 A. In contrast, narrow distance distributions were found for melittin complexed with CaM, 8.2 A, or with POPC vesicles, 4.9 A. A somewhat wider distribution was found for the melittin complex with TnC, 12.8 A, suggesting the presence of heterogeneity in the mode of binding between melittin and TnC. For all the complexes the mean Trp-19 to dansyl distance was near 20 A. This value is somewhat smaller than expected for the free alpha-helical state of melittin, suggesting that binding with CaM or TnC results in a modest decrease in the length of the melittin molecule.