Interaction of fucoidan with the proteins of the complement classical pathway

Fucoidan inhibits complement by mechanisms that so far remain to be unraveled, and the objective of this work was to delineate the mode of inhibition by this sulfated polysaccharide. For that purpose, low molecular weight fractions of algal (Ascophyllum nodosum) fucoidan containing the disaccharide unit [-->3)-alpha-L-Fuc(2SO3(-))-(1-->4)-alpha-L-Fuc(2,3diSO3(-))-(1-->](n) have been studied. Gel co-affinity electrophoresis and a new affinity capillary electrophoresis (ACE) method have been implemented to characterize fucoidan-complement protein complexes. Fucoidan binds C1q, likely to its collagen-like region through interactions involving lysine residues, and then prevents the association of the C1r(2)-C1s(2) subunit, required to form the fully active C1. In addition to C1q, fucoidan forms a complex with the protein C4 as observed by ACE. The fucoidan inhibits the first steps of the classical pathway activation that is of relevance in view of the proinflammatory effects of the subsequent products of the cascade. This study shows that a high level of inhibitory activity can be achieved with low molecular weight carbohydrate molecules and that the potential applicability of fucoidan oligosaccharides for therapeutic complement inhibition is worthy of consideration.     

On-line post-capillary affinity detection of immunoglobulin G for capillary zone electrophoresis

To address the quality issues of antibody manufacturing, post-capillary affinity detection of immunoglobulin G (IgG) is developed for capillary zone electrophoresis. In analogy to a two-dimensional separation system, capillary zone electrophoresis (CZE), as the first dimension, resolves IgG variants based on their differences in molecular structure. IgG variants separated by CZE are discriminated against other serum and cellular proteins by affinity complex formation with protein A binding fragment in a post-capillary reactor. The analytical power of post-capillary affinity detection is demonstrated for rapid and selective heterogeneity analysis of human IgG subclasses and monoclonal antibodies in complex sample matrices. By comparing with pre-capillary formation of affinity complexes between IgG and protein A, post-capillary affinity detection clearly exhibit greater resolving power for examining IgG microheterogeneity. Affinity complex formation prior to CZE analysis, however, has the advantage of lower detection limits. Detection limits suffer with post-capillary affinity detection because of the high fluorescence background contributed by the fluorescently labeled protein A in the post-capillary reactor, and the need to determine a small change in the background level upon complex formation.     

Regioselective desulfation of sulfated L-fucopyranoside by a new sulfoesterase from the marine mollusk Pecten maximus: application to the structural study of algal fucoidan (Ascophyllum nodosum).

The study of the structural bases of the biological properties of algal fucoidan (Ascophyllum nodosum) led us to look for enzymes able to modify this sulfated polysaccharide. In this context, we found a sulfoesterase activity in the digestive glands of the common marine mollusk Pecten maximus, which is active on fucoidan. This sulfoesterase activity was shown by capillary electrophoresis and 13C-1H NMR (500 MHz) analysis of the enzymatic hydrolysis of the fucoidan, of fucoidan oligosaccharides and of sulfated fucose isomers. We report the exhaustive list of all proton and carbon chemical shifts for each of the three isomers of sulfated-l-fucose (including of their alpha/beta anomers), i.e. the 2-O-, 3-O- and 4-O-sulfated fucose, which have been useful reference values for the assignments of NMR spectra of fucoidan oligosaccharides upon enzymatic desulfation. Our results demonstrated a high regioselectivity for this sulfoesterase, which hydrolyzes only the sulfate group at the 2-O position of the fucopyranoside. Therefore, this sulfoesterase is a helpful tool in the structure-activity study of the fucoidan, as the literature data suggest that the 2-O-sulfation level play a central role in the biological properties of the polysaccharide.     

Importance of lysine 125 for heparin binding and activation of antithrombin

The anticoagulant sulfated polysaccharide, heparin, binds to the plasma coagulation proteinase inhibitor, antithrombin, and activates it by a conformational change that results in a greatly increased rate of inhibition of target proteinases. Lys125 of antithrombin has previously been implicated in this binding by chemical modification and site-directed mutagenesis and by the crystal structure of a complex between antithrombin and a pentasaccharide constituting the antithrombin-binding region of heparin. Replacement of Lys125 with Met or Gln in this work reduced the affinity of antithrombin for full-length heparin or the pentasaccharide by 150-600-fold at I = 0.15, corresponding to a loss of 25-33% of the total binding energy. The affinity decrease was due both to disruption of approximately three ionic interactions, indicating that Lys125 and two other basic residues of antithrombin act cooperatively in binding to heparin, and to weakened nonionic interactions. The mutations caused a 10-17-fold decrease in the affinity of the initial, weak binding step of the two-step mechanism of heparin binding to antithrombin. They also increased the reverse rate constant of the second, conformational change step by 10-50-fold. Lys125 is thus a major heparin-binding residue of antithrombin, contributing an amount of binding energy comparable to that of Arg129, but less energy than Lys114. It is the first residue identified so far that has a critical role in the initial recognition of heparin by antithrombin, but also appreciably stabilizes the heparin-induced activated state of the inhibitor. These effects are exerted by interactions of Lys125 with the nonreducing end of the heparin pentasaccharide.     

Association of thrombin-antithrombin III complex with vitronectin in serum

Purification of vitronectin by identical procedures from serum instead of plasma results in the coisolation of an additional protein component with mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 82 kDa. We show that this component is the thrombin-antithrombin III complex based on the following evidence. Similar to a complex constructed using purified thrombin and antithrombin III, the 82-kDa component has a reduced molecular size of 69 kDa if it is not boiled prior to SDS-PAGE. Upon prolonged boiling in SDS it dissociates into 56- and 32-kDa components which co-migrate in SDS-PAGE with purified antithrombin III and thrombin, respectively. The 82- and 56-kDa components react with an antiserum against antithrombin III, and an antiserum prepared against the 82-kDa complex reacts with purified antithrombin III. Thrombin-antithrombin III complex, from either serum or recalcified clotted plasma, bound to vitronectin immobilized on Sepharose or plastic. However, purified antithrombin III which had not reacted with thrombin lacked affinity for vitronectin as did antithrombin III from citrated plasma. Purified antithrombin III acquired affinity for immobilized vitronectin if it was complexed with thrombin or was modified by radioiodination. Binding of vitronectin to antithrombin III coated on plastic was demonstrated using enzyme-linked immunosorbent assay. These results demonstrate that vitronectin binds thrombin-antithrombin III complexes through a cryptic site in antithrombin III which can be exposed when antithrombin III is radioiodinated, bound to plastic, or complexed with thrombin. Since vitronectin can interact with cells, the binding of vitronectin to the thrombin-antithrombin III complex may serve to facilitate the interaction of this complex with cell surfaces.     

Sulfated galactan is a catalyst of antithrombin-mediated inactivation of alpha-thrombin

Novel compounds presenting anticoagulant activity, such as sulfated polysaccharides, open new perspectives in medicine. Elucidation of the molecular mechanism behind this activity is desirable by itself, as well as because it allows for the design of novel compounds. In the present study, we investigated the action of an algal sulfated galactan, which potentiates alpha-thrombin inactivation by antithrombin. Our results indicate the following: 1) both the sulfated galactan and heparin potentiate protease inactivation by antithrombin at similar molar concentrations, however they differ markedly in the molecular size required for their activities; 2) this galactan interacts predominantly with exosite II on alpha-thrombin and, similar to heparin, catalyzes the formation of a covalent complex between antithrombin and the protease; 3) the sulfated galactan has a higher affinity for alpha-thrombin than for antithrombin. We propose that the preferred pathway of sulfated galactan-induced inactivation of alpha-thrombin by antithrombin starts with the polysaccharide binding to the protease through a high-affinity interaction. Antithrombin is then added to the complex and the protease is inactivated by covalent interactions. Finally, the antithrombin-alpha-thrombin covalent complex dissociates from the polysaccharide chain. This mechanism resembles the action of heparin with low affinity for antithrombin, as opposed to heparin with high affinity for serpin.     

Macromolecule-ligand binding studied by the Hummel and Dreyer method: current state of the methodology

The use of the Hummel and Dreyer method to measure binding parameters of ligand-macromolecule associations is reviewed. The possibility to determine the number of binding sites and their association constants, even in the case of low affinity, and to control the free ligand concentration as an independent variable are the main advantages of the method. The conditions of the validity are rapid equilibrium kinetics, independence between ligand binding and macromolecule association, and identical retention rates between free and bound macromolecules. Initially developed on soft gels, the method has been applied to high-performance chromatography and capillary zone electrophoresis. Technical progress such as increase in resolution, detection sensitivity, and automation have improved its utilization. The binding parameters given by the Hummel and Dreyer method are in general similar to those obtained by other techniques, in comparable experimental conditions (equilibrium dialysis, ultrafiltration, frontal elution, vacancy peak method, vacancy affinity capillary electrophoresis, retention analysis, affinity chromatography and affinity capillary electrophoresis, physical methods). The choice between these methods is directed by material availability and practical constraints. Separation by new types of chromatographic columns or by capillary zone electrophoresis would enable the study of the simultaneous binding of different drugs on the same macromolecule and their competition.     

Ultrasensitive protein-DNA binding assays

Protein-DNA binding assays have been used in a variety of fields from fundamental studies regarding the binding process itself, to serving as probes for the detection, quantification and separation of target analytes. These assays have been used for the study of protein-DNA complex stoichiometry, the detection of DNA damage, and real-time separation of free and bound complexes by electrophoretic mobility. Synthetic DNA oligonucleotides, known as aptamers, have been increasingly used for affinity binding assays to proteins, as well as for separation studies and as biosensors. Recent advances have been made in protein-DNA binding assays using capillary electrophoresis, laser-induced fluorescence, fluorescence polarization, molecular beacons, and affinity chromatography.     

Investigation of enantioselective ligand–protein binding and displacement interactions using capillary electrophoresis

When a protein such as human serum albumin is added to the separation buffer in capillary electrophoresis, the mobility of solutes which bind to that protein may be altered. The change in mobility of the solute is a function both of the strength of the binding interaction, and the difference in mobility between the free solute and protein additive. By adding other ligands which themselves bind to the protein, the strength of the solute–protein binding may be modified, leading to a measurable change in the mobility of the solute. These effects are particularly striking for chiral compounds, where enantioselectivity may be completely lost on addition of a competitive ligand. Capillary electrophoresis with human serum ablumin as a buffer additive was used to separate the enantiomers of benzoin and three phenothiazine derivatives. A comparison of the binding of (S)-benzoin to human serum albumin as determined by capillary electrophoresis and by ultrafiltration was made. A variety of other ligands were then added to the buffer along with the protein, and the effects on mobility and enantioselectivity were studied. The displacers included (R)- and (S)-oxazepam hemisuccinate, (R)- and (S)-warfarin, nitrazepam, phenylbutazone, and octanoic acid. From the results obtained, it seems that capillary electrophoresis may be a useful, rapid method to screen for drug–drug interactions. There are some advantages of using this technique to study protein–ligand interactions: Only very small amounts of ligand are needed (useful when dealing with metabolites); for chiral compounds, if protein binding is stereoselective, then the method is also stereoselective, so single enantiomers are not needed; finally, measurements are obtained in solution, without the need for immobilization of the protein. A disadvantage is that the ligand and protein must have significantly different electrophoretic mobilities. © 1994 Wiley-Liss, Inc.     

Sulfated galactan is a catalyst of antithrombin-mediated inactivation of α-thrombin

Novel compounds presenting anticoagulant activity, such as sulfated polysaccharides, open new perspectives in medicine. Elucidation of the molecular mechanism behind this activity is desirable by itself, as well as because it allows for the design of novel compounds. In the present study, we investigated the action of an algal sulfated galactan, which potentiates α-thrombin inactivation by antithrombin. Our results indicate the following: 1) both the sulfated galactan and heparin potentiate protease inactivation by antithrombin at similar molar concentrations, however they differ markedly in the molecular size required for their activities; 2) this galactan interacts predominantly with exosite II on α-thrombin and, similar to heparin, catalyzes the formation of a covalent complex between antithrombin and the protease; 3) the sulfated galactan has a higher affinity for α-thrombin than for antithrombin. We propose that the preferred pathway of sulfated galactan-induced inactivation of α-thrombin by antithrombin starts with the polysaccharide binding to the protease through a high-affinity interaction. Antithrombin is then added to the complex and the protease is inactivated by covalent interactions. Finally, the antithrombin–α-thrombin covalent complex dissociates from the polysaccharide chain. This mechanism resembles the action of heparin with low affinity for antithrombin, as opposed to heparin with high affinity for serpin.     

Computer simulations and experimental studies of gel mobility patterns for weak and strong non-cooperative protein binding to two targets on the same DNA: application to binding of tet repressor variants to multiple and single tet operator sites

A series of computer simulations of gel patterns assuming non-cooperative binding of a protein to two targets on the same DNA fragment was performed and applied to interprete gel mobility shift experiments of Tet repressor-tet operator binding. While a high binding affinity leads to the expected distribution of free DNA, DNA bound by one repressor dimer and DNA bound by two repressor dimers, a lower affinity or an increased electrophoresis time results in the loss of the band corresponding to the singly occupied complex. The doubly occupied complex remains stable under these conditions. This phenomenon is typical for protein binding to DNA fragments with two identical sites. It results from statistical disproportionation of the singly occupied complex in the gel. The lack of the singly occupied complex is commonly taken to indicate cooperative binding, however, our analysis shows clearly, that cooperativity is not needed to interprete these results. Tet repressor proteins and small DNA fragments with two tet operator sites have been prepared from four classes of tetracycline resistance determinants. The results of gel mobility shift analyses of various complexes of these compounds confirm the predictions. Furthermore, calculated gel patterns assuming different gel mobilities of the two singly occupied complexes show discrete bands only if the electrophoresis time is shorter than the inverse of the microscopic dissociation rate constant. Simulations assuming increasing dissociation rates predict that the two bands first merge into one, which then disappears. This behavior was verified by gel mobility analyses of Tet repressor-tet operator titrations at increased salt concentrations as well as by direct footprinting of the complexes in the gel. It is concluded that comparison of the intensities of the single and the double occupation bands allow a rough estimation of the dissociation rate constant. On this basis the sixteen possible Tet repressor-tet operator combinations can be ordered with decreasing binding affinities by a simple gel shift experiment. The implications of these results for gel mobility analyses of other protein-DNA complexes are discussed.     

Analysis of the interaction between hyaluronan and hyaluronan-binding proteins by capillary affinity electrophoresis: significance of hyaluronan molecular size on binding reaction

We developed a method for the analysis of the interaction between hyaluronan (HA) oligosaccharides and hyaluronan-binding proteins (HABPs) using capillary affinity electrophoresis (CAE). The method is based on high-resolution separation of fluorescent-labeled HA molecules in the presence of hyaluronan-binding proteins at different concentrations by capillary electrophoresis (CE) with laser-induced fluorescent detection. Hyaluronan-binding protein from bovine nasal cartilage interacts strongly with HA decasaccharide or larger oligosaccharides. Effect of the molecular size of HA oligomers clearly showed that longer carbohydrate chains than decasaccharide were required for recognition by HA binding protein. Interestingly, the interaction did not cause retardation of HA oligomers as observed in many binding reactions such as the interaction between pharmaceuticals and serum albumin, but showed disappearance of the oligomer peak. Although we cannot explain the accurate mechanism on the interaction, disappearance is probably due to low equilibrium rate between free and conjugate states. The present technique will be useful to compare the relative binding affinity, and to understand the mechanism on the interaction between hyaluronan and hyaluronan-binding proteins.     

Polysaccharide structural features that are critical for the binding of sulfated fucans to bindin, the adhesive protein from sea urchin sperm

We have investigated the structural features of sulfated fucose-containing polysaccharides which are responsible for their selective binding to Strongylocentrotus purpuratus bindin. The data presented demonstrate that the sulfate esters and a molecular weight in excess of approximately 15,000 are required for high affinity binding of the fucans to bindin. Desulfation destroys the binding activity of the fucans, which can be fully restored by chemical resulfation. Fucan fragments of an average molecular weight of 15,000 were nearly as active as the starting material (Mr 10(6)). The observed IC50 value for fragments of Mr congruent to 10,000 and Mr congruent to 5,000 were 1 and 2 orders of magnitude higher, respectively. The binding of fucoidan to bindin is stable in high salt (50% at 1.2 M NaCl) whereas the binding of fucoidan to DEAE-cellulose or polylysine is inhibited by the concentrations of salt normally found in sea water (50% at 0.2 and 0.5 M NaCl, respectively). This result suggests that the binding mechanism is not a simple ionic interaction and that hydrogen bonding and cooperativity may also be important determinants of the binding mechanism. We also found that polyvinyl sulfate binds to bindin with high affinity and inhibits the bindin-mediated agglutination of sea urchin eggs. The results of these investigations suggest that the spatial orientation of the sulfate esters plays a critical role in determining the selectivity of sulfated polysaccharide binding and that the polysaccharide backbone does not play a direct role in the binding mechanism.     

A comparison of the strength of binding of antithrombin III,protamine and poly(l-lysine) to heparin samples of different anicoagulant activities

The limiting concentrations, i.e., those concentrations of sodium chloride required to completely disrupt the complexes of heparin with antithrombin III, protamine and poly(l-lysine), were determined using fluorescence techniques, in order to compare the binding strengths of these complexes. From the limiting salt concentration values, poly(l-lysine)_always exhibited stronger binding to heparin of a particular anticoagulant potentcy (degree of sulphation) than did protamine. The binding strengths of both complexes decreased as the degree of sulphation of the heparin participating in the complex was reduced. In contrast, the limiting salt concentration values for complexes formed between antithrombin III and heparin did not change with either the degree of sulphation or the biological potency of the heparin samples. A low-potency heparin simply contained a smaller amount of molecules which possessed the intact antithrombin III binding site (thus being fully ‘anticoagulant active’) than a high-potency sample. Low-affinity heparin did not contain these binding sites and thus showed a low affinity for antithrombin III. High-potency heparin, being highly sulphated, possessed a higher affinity for protamine and poly(l-lysine) than for antithrombin III. However, after partial N-desulphation of heparin, the subsequent heparin-protamine complex was more weakly bound than a significant proportion of the corresponding heparin-antithrombin III complexes. These in vitro findinds may have particular relevance in relation to the clinical condition termed ‘hheparin rebound’.     

The covalent nature of the human antithrombin III--thrombin bond.

1. Cleavage of the human antithrombin III--thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labelled antithrombin III. 2. Discontinuous polyacrylamide-gel electrophoresis of the reduced dissociation fragments of the complex in the presence of sodium dodecyl sulphate reveals two antithrombin III bands that do not resolve during electrophoresis without reduction. The heavy band has the electrophoretic mobility of the native protein. The light band has an apparent mol.wt. that is approx. 4000 less than the molecular weight of native antithrombin III. 3. Treatment of the cleavage products of the complex with carboxypeptidase B yields 1 mumol of arginine, a new C-terminal amino acid, per mumol of thrombin dissociated. The results indicate that during formation of the antithrombin III--thrombin complex, the inhibitor is cleaved at an arginine--X bond; this arginine residue forms a carboxylic ester with the enzyme, while the excised polypeptide remains bound through a disulphide bridge(s).     

Nonspecific electrostatic binding characteristics of the heparin-antithrombin interaction

We evaluated the role of nonspecific electrostatic binding in the interaction of antithrombin (AT) with heparin (Hp), a paradigmatic protein-glycosaminoglycan (GAG) system. To do so, we obtained the ionic-strength dependence of the binding constant, since a common feature in protein-polyelectrolyte systems is a maximum in affinity in the ionic strength range 10 mM 相似文献   

Conversion of antithrombin from an inhibitor of thrombin to a substrate with reduced heparin affinity and enhanced conformational stability by binding of a tetradecapeptide corresponding to the P1 to P14 region of the putative reactive bond loop of the inhibitor.

A synthetic tetradecapeptide having the sequence of the region of the antithrombin chain amino-terminal to the reactive bond, i.e. comprising residues P1 to P14, was shown to form a tight equimolar complex with antithrombin. A similar complex has previously been demonstrated between alpha 1-proteinase inhibitor and the analogous peptide of this inhibitor (Schulze, A. J., Baumann, U., Knof, S., Jaeger, E., Huber, R. and Laurell, C.-B. (1990) Eur. J. Biochem. 194, 51-56). The antithrombin-peptide complex had a conformation similar to that of reactive bond-cleaved antithrombin and, like the cleaved inhibitor, also had a higher conformational stability and lower heparin affinity than intact antithrombin. These properties suggest that the peptide bound to intact antithrombin at the same site that the P1 to P14 segment of the inhibitor occupies in reactive-bond-cleaved antithrombin, i.e. was incorporated as a sixth strand in the middle of the major beta-sheet, the A sheet. The extent of complex formation was reduced in the presence of heparin with high affinity for antithrombin, which is consistent with heparin binding and peptide incorporation being linked. Antithrombin in the complex with the tetradecapeptide had lost its ability to inactivate thrombin, but the reactive bond of the inhibitor was cleaved as in a normal substrate. These observations suggest a model, analogous to that proposed for alpha 1-proteinase inhibitor (Engh, R.A., Wright, H.T., and Huber, R. (1990) Protein Eng. 3, 469-477) for the structure of intact antithrombin, in which the A sheet contains only five strands and the P1 to P14 segment of the chain forms part of an exposed loop of the protein. The results further support a reaction model for serpins in which partial insertion of this loop into the A sheet is required for trapping of a proteinase in a stable complex, and complete insertion is responsible for the conformational change accompanying cleavage of the reactive bond of the inhibitor.     

The N-terminal segment of antithrombin acts as a steric gate for the binding of heparin.

The binding of heparin causes a conformational change in antithrombin to give an increased heparin binding affinity and activate the inhibition of thrombin and factor Xa. The areas of antithrombin involved in binding heparin and stabilizing the interaction in the high-affinity form have been partially resolved through the study of both recombinant and natural variants. The role of a section of the N-terminal segment of antithrombin, residues 22-46 (segment 22-46), in heparin binding was investigated using rapid kinetic analysis of the protein cleaved at residues 29-30 by limited proteolysis with thermolysin. The cleaved antithrombin had 5.5-fold lowered affinity for heparin pentasaccharide and 1.8-fold for full-length, high-affinity heparin. It was shown that, although the initial binding of heparin is slightly enhanced by the cleavage, it dissociates much faster from the cleaved form, giving rise to the overall decrease in heparin affinity. This implies that the segment constituting residues 22-46 in the N terminus of antithrombin hinders access to the binding site for heparin, hence the increased initial binding for the cleaved form, whereas, when heparin is bound, segment 22-46 is involved in the stabilization of the binding interaction, as indicated by the increased dissociation constant. When the heparin pentasaccharide is bound to antithrombin prior to incubation with thermolysin, it protects the N-terminal cleavage site, implying that segment 22-46 moves to interact with heparin in the conformational change and thus stabilizes the complex.     

The effect of fucoidan on tyrosinase: computational molecular dynamics integrating inhibition kinetics

Fucoidan is a complex sulfated polysaccharide extracted from brown seaweed and has a wide variety of biological activities. In this study, we investigated the inhibitory effect of fucoidan on tyrosinase via a combination of inhibition kinetics and computational simulations. Fucoidan reversibly inhibited tyrosinase in a mixed-type manner. Time-interval kinetics showed that the inhibition was processed as first order with biphasic processes. For further insight, we simulated dockings with various sizes of molecular models (monomer to decamer) of fucoidan and showed that the best binding energy change results were obtained from the pentamer (?1.89?kcal/mol) and the hexamer (?1.97?kcal/mol) models of AutoDock Vina. The molecular dynamics simulation confirmed the binding mechanisms between tyrosinase and fucoidan and suggested that fucoidan mostly interacts with several residues including copper ions located in the active site. Our study suggests that fucoidan might be a potential natural antipigment agent.     

Calcium-dependent and -independent binding of soybean calmodulin isoforms to the calmodulin binding domain of tobacco MAPK phosphatase-1

The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca(2+) signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca(2+) with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca(2+), the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca(2+)-loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided.