The interaction of calmodulin with melittin

Studies utilizing the interaction of melittin with the 1-106 fragment of calmodulin, the protection of calmodulin from tryptic digestion by melittin, and the interaction of the carbocyanine dye Stains-all with the calmodulin-melittin complex have indicated that complex formation of calmodulin with melittin involves the alpha-helical connecting bridge joining the N- and C-terminal lobes of calmodulin.     

The interaction of delta-hemolysin with calmodulin

Delta-Hemolysin forms a 1:1 complex with Ca2+ -liganded calmodulin. Probably because of the pronounced tendency of delta-hemolysin to self-associate, the apparent binding affinity is much less than that for melittin. Complex formation is reflected by an increase in quantum yield of Trp-15 of delta-hemolysin and by increased shielding from acrylamide quenching. There is, however, no indication of a change in peptide molecular ellipticity. The binding of 2-toluidinyl-naphthalene-6-sulfonate is reduced by complex formation, suggesting the involvement of a hydrophobic region. Complex formation also blocks the proteolysis by trypsin of the bond between residues 77 and 78. The time decays of fluorescence intensity and anisotropy for tryptophan are multiexponential for both free and complexed delta-hemolysin; the average decay time for intensity is substantially increased for the complex. The localized mobility of tryptophan is greatly reduced in the complex. Complex formation appears to involve both the C-terminal lobe and the connecting strand of calmodulin.     

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.     

The interaction of calmodulin with amphiphilic peptides

Calmodulin has recently been shown to form exceptionally tight, calcium-dependent complexes with several natural peptides (Kdiss greater than 10(-7) M). These peptides were demonstrated to be capable of forming basic, amphiphilic alpha-helices. To further illustrate the importance of this structural feature for calmodulin binding, several other amphiphilic alpha-helical peptides were tested for their ability to bind calmodulin. To monitor complexes of high affinity (greater than 10(8) M-1), a new competition assay was devised with Sepharose 4B-conjugated melittin. Stoichiometries were assessed by electrophoresis and equilibrium size exclusion chromatography. Three peptides, which were designed to form idealized amphiphilic alpha-helices were tested. The basic peptides, N alpha-9-fluorenylmethoxycarboxyl-(FMOC)-(Leu-Lys-Lys-Leu-Leu-Lys-L eu)1 and FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu)2 bind calmodulin in a 1:1 complex with dissociation constants of 150 and 3 nM, respectively. The acidic peptide, FMOC-(Leu-Glu-Glu-Leu-Leu-Glu-Leu)2 failed to bind calmodulin, even at micromolar concentrations. Complex formation between calmodulin and the 14-residue basic peptide leads to an increase in the helicity of the complex which is attributed to an increase of about 50% in the helicity of the peptide. Calmodulin also interacts with the neutral alpha-helical peptide toxin delta-hemolysin. Concomitant with binding, the fluorescence maximum of the unique Trp residue increases 2-fold and is blue-shifted. A dissociation constant could not be unambiguously estimated though, since delta-hemolysin has a strong tendency to self-aggregate. The above data support our hypothesis that a basic, amphiphilic alpha-helix is a structural feature which underlies the calmodulin-binding properties common to a variety of peptides.     

AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation.

The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.     

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.     

The interaction of calmodulin with erythrocyte membrane proteins

The method of sedimentation equilibrium in an air-driven ultracentrifuge (Airfuge) has been employed to investigate the interaction of 125I-calmodulin with the cytoskeletal components of the human red cell membrane. The results indicate significant calcium-dependent calmodulin binding activity in the low and high ionic strength extracts of the human erythrocyte membrane. The interaction of 125I-calmodulin with the low ionic strength extract proteins is analysed quantitatively. Further purification of the high ionic strength extract comprising mainly band 2.1 and band 4.1 results in the elution of calmodulin binding activity in a purified fraction of band 4.1.     

The intermolecular interaction between the PH domain and the C-terminal domain of Arabidopsis dynamin-like 6 determines lipid binding specificity

Dynamin and its related proteins are a group of mechanochemical proteins involved in the modulation of lipid membranes in various biological processes. Here we investigate the nature of membrane binding of the Arabidopsis dynamin-like 6 (ADL6) involved in vesicle trafficking from the trans-Golgi network to the central vacuole. Fractionation experiments by continuous sucrose gradients and gel filtration revealed that the majority of ADL6 is associated with membranes in vivo. Amino acid sequence analysis revealed that ADL6 has a putative pleckstrin homology (PH) domain. In vitro lipid binding assays demonstrated that ADL6 showed high affinity binding to phosphatidylinositol 3-phosphate (PtdIns-3-P) and that the PH domain was responsible for this interaction. However, the PH domain alone binds equally well to both PtdIns-3-P and phosphatidylinositol 4-phosphate (PtdIns-4-P). Interestingly, the high affinity binding of the PH domain to PtdIns-3-P was restored by a protein-protein interaction between the PH domain and the C-terminal region. In addition, deletion of the inserted regions within the PH domain results in high affinity binding of the PH domain to PtdIns-3-P. These results suggest that ADL6 binds specifically to PtdIns-3-P and that the lipid binding specificity is determined by the interaction between the PH domain and the C-terminal domain of ADL6.     

The interaction of thrombin with fibrinogen. A structural basis for its specificity.

The structure of the ternary complex of human alpha-thrombin with a covalently bound analogue of fibrinopeptide A and a C-terminal hirudin peptide has been determined by X-ray diffraction methods at 0.25 nm resolution. Fibrinopeptide A folds in a compact manner, bringing together hydrophobic residues that slot into the apolar binding site of human alpha-thrombin. Fibrinogen residue Phe8 occupies the aryl-binding site of thrombin, adjacent to fibrinogen residues Leu9 and Val15 in the S2 subsite. The species diversity of fibrinopeptide A is analysed with respect to its conformation and its interaction with thrombin. The non-covalently attached peptide fragment hirudin(54-65) exhibits an identical conformation to that observed in the hirudin-thrombin complex. The occupancy of the secondary fibrinogen-recognition exosite by this peptide imposes restrictions on the manner of fibrinogen binding. The surface topology of the thrombin molecule indicates positions P1'-P3', differ from those of the canonical serine-proteinase inhibitors, suggesting a mechanical model for the switching of thrombin activity from fibrinogen cleavage to protein-C activation on thrombomodulin complex formation. The multiple interactions between thrombin and fibrinogen provide an explanation for the narrow specificity of thrombin. Structural grounds can be put forward for certain congenital clotting disorders.     

The structural basis of actin interaction with multiple WH2/beta-thymosin motif-containing proteins

Participation of actin in cellular processes relies on the dynamics of filament assembly. Filament elongation is fed by monomeric actin in complex with either profilin or a Wiscott-Aldrich syndrome protein (WASP) homology domain 2 (WH2)/beta-thymosin (betaT) domain. WH2/betaT motif repetition (typified by ciboulot) or combination with nonrelated domains (as found in N-WASP) results in proteins that yield their actin to filament elongation. Here, we report the crystal structures of actin bound hybrid proteins, constructed between gelsolin and WH2/betaT domains from ciboulot or N-WASP. We observe the C-terminal half of ciboulot domain 2 bound to actin. In solution, we show that cibolout domains 2 and 3 bind to both G- and F-actin, and that whole ciboulot forms a complex with two actin monomers. In contrast, the analogous portion of N-WASP WH2 domain 2 is detached from actin, indicating that the C-terminal halves of the betaT and WH2 motifs are not functionally analogous.     

A structural basis for the interaction of urea with lysozyme.

The effect of urea on the crystal structure of hen egg-white lysozyme has been investigated using X-ray crystallography. High resolution structures have been determined from crystals grown in the presence of 0, 0.7, 2, 3, 4, and 5 M urea and from crystals soaked in 9 M urea. All the forms are essentially isomorphous with the native type II crystals, and the derived structures exhibit excellent geometry and RMS differences from ideality in bond distances and angles. Comparison of the urea complex structures with the native enzyme (type II form, at 1.5 A resolution) indicates that the effect of urea is minimal over the concentration range studied. The mean difference in backbone conformation between the native enzyme and its urea complexes varies from 0.18 to 0.49 A. Conformational changes are limited to flexible surface loops (Thr 69-Asn 74, Ser 100-Asn 103), the active site loop (Asn 59-Cys 80), and the C-terminus (Cys 127-Leu 129). Urea molecules are bound to distinct sites on the surface of the protein. One molecule is bound to the active site cleft's C subsite, at all concentrations, in a fashion analogous to that of the N-acetyl substituent of substrate and inhibitor sugars normally bound to this site. Occupation of this subsite by urea alone does not appear to induce the conformational changes associated with inhibitor binding.     

The structural basis for autoinhibition of FLT3 by the juxtamembrane domain

FLT3 is a type III receptor tyrosine kinase that is thought to play a key role in hematopoiesis. Certain classes of FLT3 mutations cause constitutively activated forms of the receptor that are found in significant numbers of patients with acute myelogenous leukemia (AML). The mutations occur either in the activation loop, for example, as point mutations of Asp835 or as internal tandem duplication (ITD) sequences in the juxtamembrane (JM) domain. To further understand the nature of FLT3 autoinhibition and regulation, we have determined the crystal structure of the autoinhibited form of FLT3. This structure shows the autoinhibitory conformation of a complete JM domain in this class of receptor tyrosine kinases. The detailed inhibitory mechanism of the JM domain is revealed, which is likely utilized by other members of type III receptor tyrosine kinases.     

The interaction of cyclosporin and calmodulin

The interaction of cyclosporin A and dansyl cyclosporin A with bovine and wheat germ calmodulin has been monitored by measurements of induced changes in dansyl and bound toluidinyl naphthalene sulfonate fluorescence. The interaction is Ca2(+)-dependent and 1:1. Measurements of the efficiency of radiationless energy transfer from bound dansyl cyclosporin A to an acceptor group located on Cys-27 of wheat germ calmodulin suggest that the primary binding site is not located on the N-terminal lobe (residues 1-65). However, studies with proteolytic fragments of calmodulin indicate that elements of the N-terminal half-molecule (residues 1-77) may be involved in the stabilization of the binding site. The binding of cyclosporin alters the physical properties of calmodulin and, in particular, reduces the localized rotational mobility of a fluorescent probe.     

The structural basis for the oligomerization of the N-terminal domain of SATB1

Special AT-rich sequence-binding protein 1 (SATB1) is a global chromatin organizer and gene expression regulator essential for T-cell development and breast cancer tumor growth and metastasis. The oligomerization of the N-terminal domain of SATB1 is critical for its biological function. We determined the crystal structure of the N-terminal domain of SATB1. Surprisingly, this domain resembles a ubiquitin domain instead of the previously proposed PDZ domain. Our results also reveal that SATB1 can form a tetramer through its N-terminal domain. The tetramerization of SATB1 plays an essential role in its binding to highly specialized DNA sequences. Furthermore, isothermal titration calorimetry results indicate that the SATB1 tetramer can bind simultaneously to two DNA targets. Based on these results, we propose a molecular model whereby SATB1 regulates the expression of multiple genes both locally and at a distance.     

The structural basis of localization and signaling by the focal adhesion targeting domain

The localization of focal adhesion kinase (FAK) to sites of integrin clustering initiates downstream signaling. The C-terminal focal adhesion targeting (FAT) domain causes this localization by interacting with talin and paxillin. FAT also mediates signaling through Grb2 via phosphorylated Y925. We report two crystal structures of the FAT domain. Large rearrangements of the structure are indicated to allow phosphorylation of Y925 and subsequent interaction with Grb2. Sequence homology and structural compatibility suggest a FAT-like fold for the C-terminal domains of CAS, Efs/Sin, and HEF1. A structure-based alignment including these proteins and the vinculin tail domain reveals a conserved region that could play a role in focal adhesion targeting. Previously postulated "paxillin binding subdomains" may contribute to structural integrity rather than directly to paxillin binding.     

Conformation of the calmodulin-binding domain of metabotropic glutamate receptor subtype 7 and its interaction with calmodulin

Calmodulin (CaM), a Ca(2+)-binding protein, is a well-known regulator of various cellular functions. One of the targets of CaM is metabotropic glutamate receptor 7 (mGluR7), which serves as a low-pass filter for glutamate in the pre-synaptic terminal to regulate neurotransmission. Surface plasmon resonance (SPR), circular dichroism (CD) spectroscopy and nuclear magnetic spectroscopy (NMR) were performed to study the structure of the peptides corresponding to the CaM-binding domain of mGluR7 and their interaction with CaM. Unlike well-known CaM-binding peptides, mGluR7 has a random coil structure even in the presence of trifluoroethanol. Moreover, NMR data suggested that the complex between Ca(2+)/CaM and the mGluR7 peptide has multiple conformations. The mGluR7 peptide has been found to interact with CaM even in the absence of Ca(2+), and the binding is directed toward the C-domain of apo-CaM rather than the N-domain. We propose a possible mechanism for the activation of mGluR7 by CaM. A pre-binding occurs between apo-CaM and mGluR7 in the resting state of cells. Then, the Ca(2+)/CaM-mGluR7 complex is formed once Ca(2+) influx occurs. The weak interaction at lower Ca(2+) concentrations is likely to bind CaM to mGluR7 for the fast complex formation in response to the elevation of Ca(2+) concentration.     

The interaction of melittin with calmodulin and its tryptic fragments

Melittin has been found to interact with both the N- and C-terminal half-molecules of calmodulin, as well as the intact molecule, in the presence of Ca2+. The interaction results in a major change in the microenvironment of Trp-19, which is in a more nonpolar, solvent-shielded, and immobilized microenvironment in the complex. The properties of Tyr-99 and Tyr-138 of calmodulin are altered by complex formation. From measurements of the efficiencies of radiationless energy transfer from Trp-19 to the nitro derivatives of Tyr-99 and/or Tyr-138, it is concluded that Trp-19 is located in proximity to the C-terminal lobe of calmodulin in the complex.     

Yeast calmodulin: structural and functional differences compared with vertebrate calmodulin

Calmodulin of the baker's yeast (Saccharomyces cerevisiae) showed a similar affinity for Ca2+ to that of vertebrate calmodulin. The maximum binding number of Ca2+ to yeast calmodulin was, however, 3 mol/mol, which is lower than that of vertebrate calmodulin (4 mol/mol). The same maximum activity of porcine brain phosphodiesterase was attained when 100 times higher concentration of yeast calmodulin than that of vertebrate calmodulin was added. On the other hand, the maximum activation of chicken gizzard myosin light chain kinase was attained with 1,000 times higher concentration of yeast calmodulin than that of vertebrate calmodulin, and the maximum activity with yeast calmodulin was less than 1/5 of that with vertebrate calmodulin. Several amino acid substitutions observed in the yeast calmodulin, particularly at the alpha-helical rod connecting the two globular domains, may affect the interaction mode of various target enzymes with this calmodulin.     

Microarray analysis of Akt PH domain binding employing synthetic biotinylated analogs of all seven phosphoinositide headgroup isomers

Signaling lipids control many of the most important biological pathways, typically by recruiting cognate protein binding targets to cell surfaces, thereby regulating both their function and subcellular localization. A critical family of signaling lipids is that of the phosphatidylinositol polyphosphates (PIP(n)s), which is composed of seven isomers that vary based on phosphorylation pattern. A key protein that is activated upon PIP(n) binding is Akt, which then plays important roles in regulating the cell cycle, and is thus aberrant in disease. Characterization of protein-PIP(n) binding interactions is hindered by the complexity of the membrane environment and of the PIP(n) structures. Herein, we describe two rapid assays of use for characterizing protein-PIP(n) binding interactions. First, a microplate-based binding assay was devised to characterize the binding of effectors to immobilized synthetic PIP(n) headgroup-biotin conjugates corresponding to all seven isomers. The assay was implemented for simultaneous analysis of Akt-PH domain, indicating PI(3,4,5)P(3) and PI(3,4)P(2) as the primary ligands. In addition, density-dependant studies indicated that the amount of ligand immobilized on the surface affected the amplitude of protein binding, but not the affinity, for Akt-PH. Since the PIP(n) ligand motifs used in this analysis lack the membrane environment and glycerolipid backbone, yet still exhibit high-affinity protein binding, these results narrow down the structural requirements for Akt recognition. Additionally, binding detection was also achieved through microarray analysis via the robotic pin printing of ligands onto glass slides in a miniaturized format. Here, fluorescence-based detection provided sensitive detection of binding using minimal amounts of materials. Due to their high-throughput and versatile attributes, these assays provide invaluable tools for probing and perturbing protein-membrane binding interactions.     

Calcium-dependent interaction of transducin with calmodulin Sepharose

Affinity chromatography on calmodulin Sepharose showed that transducin, the G protein of bovine retinal rod outer segments, interacts with the Ca²⁺-calmodulin complex. This may mean that in the dark, rod outer segment calmodulin is largely in the bound state. It was assumed that photoactivation of rods induces a change in the calmodulin concentration in the cytoplasm of rod outer segments and this may be one of the processes leading to light adaptation of the photoreceptor.