Spectroscopic studies reveal details of substrate-induced conformational changes distant from the active site in isopenicillin N synthase

Isopenicillin N synthase (IPNS) catalyzes formation of the β-lactam and thiazolidine rings of isopenicillin N from its linear tripeptide l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) substrate in an iron- and dioxygen (O₂)-dependent four-electron oxidation without precedent in current synthetic chemistry. Recent X-ray free-electron laser studies including time-resolved serial femtosecond crystallography show that binding of O₂ to the IPNS–Fe(II)–ACV complex induces unexpected conformational changes in α-helices on the surface of IPNS, in particular in α3 and α10. However, how substrate binding leads to conformational changes away from the active site is unknown. Here, using detailed ¹⁹F NMR and electron paramagnetic resonance experiments with labeled IPNS variants, we investigated motions in α3 and α10 induced by binding of ferrous iron, ACV, and the O₂ analog nitric oxide, using the less mobile α6 for comparison. ¹⁹F NMR studies were carried out on singly and doubly labeled α3, α6, and α10 variants at different temperatures. In addition, double electron–electron resonance electron paramagnetic resonance analysis was carried out on doubly spin-labeled variants. The combined spectroscopic and crystallographic results reveal that substantial conformational changes in regions of IPNS including α3 and α10 are induced by binding of ACV and nitric oxide. Since IPNS is a member of the structural superfamily of 2-oxoglutarate-dependent oxygenases and related enzymes, related conformational changes may be of general importance in nonheme oxygenase catalysis.     

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [³⁵S]GTPγS functional assays were performed in the presence of putative δ₁-(DPDPE: agonist, BNTX: antagonist), δ₂-(agonist: deltorphin II, Ile^5,6-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ₂- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K_D = 0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [³H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [³H]Ile^5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49 ± 0.06 and 0.30 ± 0.01 nM potency against DPDPE and deltorphin II in the [³⁵S]GTPγS functional assay, respectively. The rank order of potency of putative δ₁- and δ₂-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ₁- and δ₂-opioid receptors could not be unequivocally distinguished in vitro.     

Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP₂) to phosphoinositide-3,4,5-trisphosphate (PIP₃), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP₂, a membrane lipid.     

Dual roles of a conserved pair, Arg23 and Ser20, in recognition of multiple substrates in α-aminoadipate aminotransferase from Thermus thermophilus

To clarify the mechanism for substrate recognition of α-aminoadipate aminotransferase (AAA-AT) from Thermus thermophilus, the crystal structure of AAA-AT complexed with N-(5′-phosphopyridoxyl)-l-glutamate (PPE) was determined at 1.67 Å resolution. The crystal structure revealed that PPE is recognized by amino acid residues the same as those seen in N-(5′-phosphopyridoxyl)-l-α-aminoadipate (PPA) recognition; however, to bind the γ-carboxyl group of Glu at a fixed position, the Cα atom of the Glu moiety moves 0.80 Å toward the γ-carboxyl group in the PPE complex. Markedly decreased activity for Asp can be explained by the shortness of the aspartyl side chain to be recognized by Arg23 and further dislocation of the Cα atom of bound Asp. Site-directed mutagenesis revealed that Arg23 has dual functions for reaction, (i) recognition of γ (δ)-carboxyl group of Glu (AAA) and (ii) rearrangement of α2 helix by changing the interacting partners to place the hydrophobic substrate at the suitable position.     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by Y_Z. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[−Mn]) membranes. The rate of Mn(II) oxidation saturates at ≥10 μM in PSII(−Mn) membranes and ≥500 μM in EDC-treated PSII (−Mn) samples. Intact PSII(−Mn) membranes have only one site for Mn(II) oxidation via Y_Z (dissociation constant, K_d = 0.64 μM), while EDC-treated PSII(−Mn) samples have two sites (K_d = 1.52 and 22 μM; the latter is the low-affinity site). When PSII(−Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (K_d = 2.3 μM) for exogenous Mn(II) oxidation by Y_Z radical. In this case, the rate of electron donation via Y_Z saturated at a Mn(II) concentration ≥15 μM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HA_Z site is protected from the action of the modifier by the iron cations blocking the HA_Z site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HA_Z site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

Functional studies with membrane-bound and detergent-solubilized α2-adrenergic receptors expressed in Sf9 cells

A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery.We have expressed the three subtypes of α₂-adrenergic receptor (α₂-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of α_2B-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K_d values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that α₂-AR is active even in a lipid-free environment. The K_d values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of α₂-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads.     

New metal complexes of N2S2 tetradentate ligands: Synthesis and spectral studies

New tetradentate ligands 2-(2-mercaptoethylthio)-N-(pyridin-2-ylmethyl)acetamide H₂L¹ and 2-chloro-2-(2-mercaptoethylthio)-N-(pyridin-2-ylmethyl)acetamide H₂L² were synthesised from the reaction of 2-aminomethanepyridine with 1,4-dithian-2-one and 3-chloro-1,4-dithian-2-one, respectively. Monomeric complexes of these ligands, of general formulae K[Cr^III(Lⁿ)Cl₂], K₂[Mn^II(Lⁿ)Cl₂] and [M(Lⁿ)] (M = Fe(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II) or Hg(II); n = 1, 2) are reported. The mode of bonding and overall geometry of the complexes were determined through IR, UV-Vis, NMR and mass spectral studies, magnetic moment measurements, elemental analysis, metal content and conductance. These studies revealed octahedral geometries for the Cr(III), Mn(II) complexes, square planar for Ni(II) and Cu(II) complexes and tetrahedral for the Fe(II), Co(II), Zn(II), Cd(II) and Hg(II) complexes. The study of complex formation via molar ratio in DMF solution has been investigated and results were consistent to those found in the solid complexes with a ratio of (M:L) as (1:1).     

Metabolism of 1α,25-dihydroxyvitamin D2 by human CYP24A1

The metabolism of 1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂) by human CYP24A1 was examined using the recombinant enzyme expressed in Escherichia coli cells. HPLC analysis revealed that human CYP24A1 produces at least 10 metabolites, while rat CYP24A1 produces only three metabolites, indicating a remarkable species-based difference in the CYP24A1-dependent metabolism of 1α,25(OH)₂D₂ between humans and rats. LC-MS analysis and periodate treatment of the metabolites strongly suggest that human CYP24A1 converts 1α,25(OH)₂D₂ to 1α,24,25,26(OH)₄D₂, 1α,24,25,28(OH)₄D₂, and 24-oxo-25,26,27-trinor-1α(OH)D₂ via 1α,24,25(OH)₃D₂. These results indicate that human CYP24A1 catalyzes the C₂₄-C₂₅ bond cleavage of 1α,24,25(OH)₂D₂, which is quite effective in the inactivation of the active form of vitamin D₂. The combination of hydroxylation at multiple sites and C-C bond cleavage could form a large number of metabolites. Our findings appear to be useful to predict the metabolism of vitamin D₂ and its analogs in the human body.     

Alteration of molecular conformations and spectral properties for nickel(II), zinc(II) and copper(II) complexes having 3,8-di(thiophen-2′,2′′-yl)-1,10-phenanthroline ligands

Four transition metal complexes of 3,8-di(thiophen-2′,2″-yl)-1,10-phenanthroline (dtphen), formulated as [Ni(dtphen)₂(H₂O)₂]·(ClO₄)₂ (1), [Zn(dtphen)₂(H₂O)]·(ClO₄)₂ (2) [Cu(dtphen)₂(H₂O)]·(ClO₄)₂ (3), [Cu(dtphen)(phen)₂]·(ClO₄)₂ (4) (phen = 1,10-phenanthroline) with different metal-to-ligand ratios, were synthesized and characterized herein. The X-ray single-crystal diffraction studies of 1-4 exhibit that different molecular configurations for the dtphen ligand can be observed where the side thiophene rings adopt the trans/trans, trans/cis, trans/disorder and cis/cis conformations relative to the central 1,10-phenanthroline unit in different compounds. Fluorescence emission spectra of 1-4 in methanol show that the fluorescence emission of 2 is much stronger than the other three metal complexes, which is mainly due to its full d¹⁰ electronic configuration of Zn(II) ion.     

1α,25-Dihydroxyvitamin D3-3β-bromoacetate, a potential cancer therapeutic agent: synthesis and molecular mechanism of action

Synthesis of 1α,25-dihydroxyvitamin D₃-3β-bromoacetate (1,25(OH)₂D₃-3-BE), a potential anti-cancer agent is presented. We also report that mechanism of action of 1,25(OH)₂D₃-3-BE may involve reduction of its catabolism, as evidenced by the reduced and delayed expression of 1α,25-dihydroxyvitamin D₃-24-hydroxylase (CYP24) gene in cellular assays.     

Unexpected dependence on pH of NO release from Paracoccus pantotrophus cytochrome cd1

A previous study of nitrite reduction by Paracoccus pantotrophus cytochrome cd₁ at pH 7.0 identified early reaction intermediates. The c-heme rapidly oxidised and nitrite was reduced to NO at the d₁-heme. A slower equilibration of electrons followed, forming a stable complex assigned as 55% cFe(III)d₁Fe(II)-NO and 45% cFe(II)d₁Fe(II)-NO⁺. No catalytically competent NO release was observed. Here we show that at pH 6.0, a significant proportion of the enzyme undergoes turnover and releases NO. An early intermediate, which was previously overlooked, is also identified; enzyme immediately following product release is a candidate. However, even at pH 6.0 a considerable fraction of the enzyme remains bound to NO so another component is required for full product release. The kinetically stable product formed at the end of the reaction differs significantly at pH 6.0 and 7.0, as does its rate of formation; thus the reaction is critically dependent on pH.     

Structural and magnetic characterization of a novel series of dinuclear Cu(II) complexes bridging by 2,5-pyrazine dicarboxylate

A new series of dinuclear 2,5-pyrazine dicarboxylato-bridged copper(II) complexes were synthesized and characterized by spectroscopic techniques. The complexes have the general structural formula [Cu₂(L)₂(μ-pyzdc)](ClO₄)₂·nH₂O where L = TPA, n = 2 (1); L = pmedien, n = 2 (2); L = aepn, n = 3 (3); L = dpt, n = 2 (4); L = Medpt, n = 0 (5); L = dien, n = 0 (6) and L = MeDPA, n = 2 (7) with TPA = tris(2-pyridylmethyl)amine, pmdien = N,N,N′,N′′,N′′-pentamethyldiethylenetriamine, aepn = N-(2-aminoethyl)-1,3-diaminopropane, dpt = dipropylene-triamine, Medpt = 3,3′-diamino-N-methyldipropylamine, dien = diethylenetriamine, MeDPA = N,N-di(2-pyridylmethyl)methylamine. In these complexes, the bridging nature of the 2,5-pyrazine dicarboxylato ligand (pyzdc) was confirmed by single-crystal X-ray crystallography. The structure of the TPA complex 1 consists of μ-pyzdc bridging two Cu(II) centers in a bis(monodentate) bonding fashion through a single oxygen atom supplied by each carboxylate group of the bridged pyzdc in a distorted trigonal bipyramidal geometry achieved by the four nitrogen atoms from the TPA ligand. In the complexes 2-5 derived from tridentate amines, the bridged pyzdc acts as a bis(bidentate) ligand in a distorted square pyramidal geometry achieved by one nitrogen and one carboxylate-oxygen of pyzdc, and by the three N-atoms of the amine coligands. The intradimer Cu?Cu distances in the complexes 2-5 are in the range 6.97-7.45 ? and in it is 10.96 ? in 1. The corresponding intermolecular distances are even shorter (5.34-7.99 ?). The susceptibility measurements at variable temperatures over the 5-300 K range reveal weak antiferromagnetic coupling with J values ranging from −0.61 to −4.78 cm⁻¹.     

The dormancy-breaking stimuli “chilling,hypoxia and cyanamide exposure” up-regulate the expression of α-amylase genes in grapevine buds

It has been suggested that respiratory stress is involved in the mechanism underlying the dormancy-breaking effect of hydrogen cyanamide (H₂CN₂) and sodium azide in grapevine buds; indeed, reductions in oxygen levels (hypoxia) and inhibitors of respiration promote bud-break in grapevines. In this study, we showed that, hypoxia increased starch hydrolysis soluble sugar consumption and up-regulated the expression of α-amylase genes (Vvα-AMYs) in grapevine buds, suggesting that these biochemical changes induced by hypoxia, may play a relevant role in the release of buds from endodormancy (ED). Three of the four Vvα-AMY genes that are expressed in grapevine buds were up-regulated by hypoxia and a correlation between changes in sugar content and level of Vvα-AMY gene expression during the hypoxia treatment was found, suggesting that soluble sugars mediate the effect of hypoxia on Vvα-AMY gene expression. Exogenous applications of soluble sugars and sugar analogs confirmed this finding and revealed that osmotic stress induces the expression of Vvα-AMY1 and Vvα-AMY3 and that soluble sugars induces Vvα-AMY2 and Vvα-AMY4 gene expression. Interestingly, the plant hormone gibberellic acid (GA₃) induced the expression of Vvα-AMY3 and Vvα-AMY4 genes, while dormancy breaking stimuli, chilling and cyanamide exposure, mainly induced the expression of Vvα-AMY1 and Vvα-AMY2 genes, suggesting that these two α-amylase genes might be involved in the release of grapevine buds from the ED.     

Covalent labeling of nuclear vitamin D receptor with affinity labeling reagents containing a cross-linking probe at three different positions of the parent ligand: Structural and biochemical implications

Structure-functional characterization of vitamin D receptor (VDR) requires identification of structurally distinct areas of VDR-ligand-binding domain (VDR-LBD) important for biological properties of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). We hypothesized that covalent attachment of the ligand into VDR-LBD might alter ‘surface structure’ of that area influencing biological activity of the ligand. We compared anti-proliferative activity of three affinity alkylating derivatives of 1,25(OH)₂D₃ containing an alkylating probe at 1,3 and 11 positions. These compounds possessed high-affinity binding for VDR; and affinity labeled VDR-LBD. But, only the analog with probe at 3-position significantly altered growth in keratinocytes, compared with 1,25(OH)₂D₃. Molecular models of these analogs, docked inside VDR-LBD tentatively identified Ser237 (helix-3: 1,25(OH)₂D₃-1-BE), Cys288 (β-hairpin region: 1,25(OH)₂D₃-3-BE,) and Tyr295 (helix-6: 1,25(OH)₂D₃-11-BE,) as amino acids that are potentially modified by these reagents. Therefore, we conclude that the β-hairpin region (modified by 1,25(OH)₂D₃-3-BE) is most important for growth inhibition by 1,25(OH)₂D₃, while helices 3 and 6 are less important for such activity.     

Inhibition of the calcium-activated chloride current in cardiac ventricular myocytes by N-(p-amylcinnamoyl)anthranilic acid (ACA)

N-(p-amylcinnamoyl)anthranilic acid (ACA), a phospholipase A₂ (PLA₂) inhibitor, is structurally-related to non-steroidal anti-inflammatory drugs (NSAIDs) of the fenamate group and may also modulate various ion channels. We used the whole-cell, patch-clamp technique at room temperature to investigate the effects of ACA on the Ca²⁺-activated chloride current (I_Cl(Ca)) and other chloride currents in isolated pig cardiac ventricular myocytes. ACA reversibly inhibited I_Cl(Ca) in a concentration-dependent manner (IC₅₀ = 4.2 μM, n_Hill = 1.1), without affecting the L-type Ca²⁺ current. Unlike ACA, the non-selective PLA₂ inhibitor bromophenacyl bromide (BPB; 50 μM) had no effect on I_Cl(Ca). In addition, the analgesic NSAID structurally-related to ACA, diclofenac (50 μM) also had no effect on I_Cl(Ca), whereas the current in the same cells could be suppressed by chloride channel blockers flufenamic acid (FFA; 100 μM) or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS;100 μM). Besides I_Cl(Ca), ACA (50 μM) also suppressed the cAMP-activated chloride current, but to a lesser extent. It is proposed that the inhibitory effects of ACA on I_Cl(Ca) are PLA₂-independent and that the drug may serve as a useful tool in understanding the nature and function of cardiac anion channels.     

Experimental Evidence for the Existence of a Stable Half-Barrel Subdomain in the (β/α)8-Barrel Fold

The (β/α)₈-barrel is one of the most common folds functioning as enzymes. The emergence of two (β/α)₈-barrel enzymes involved in histidine biosynthesis, each of which has a twofold symmetric structure, has been proposed to be a consequence of tandem duplication and fusion of a (β/α)₄-half-barrel. However, little evidence has been found for the existence of an ancestral half-barrel in the evolution of other (β/α)₈-barrel proteins. In order to detect remnants of an ancestral half-barrel in the (β/α)₈-barrel structure of Escherichia coli N-(5′-phosphoribosyl)anthranilate isomerase, we engineered three potential half-barrel units, (β/α)_1-4, (β/α)_3-6, and (β/α)_5-8. Among these three arrangements, only (β/α)_3-6 is stable; it exists in equilibrium between monomeric and dimeric forms. Thus, the central segment of N-(5′-phosphoribosyl)anthranilate isomerase from E. coli can serve as a half-barrel precursor. A tandem duplication of (β/α)_3-6 yielded predominantly monomeric structures that were quite stable. This result exemplified that the structural characteristics of noncovalently assembled half-barrels could be improved by duplication and fusion. Moreover, our results may provide information regarding the local structural units that encompass interactions important for the early folding events of this ubiquitous protein conformation.     

New polymeric thiocyanatoiron(II) complex with N,N′-diethylnicotinamide - Synthesis, structure, magnetic and spectral properties

The iron(II) compound of formula [Fe(NCS)₂(dena)₂]_n (dena = N,N′-diethylnicotinamide) has been prepared by the reaction between iron(III) thiocyanate and dena in ethanol solution. The complex was characterized by elemental analysis, spectral and magnetic measurements. Single-crystal X-ray diffraction methods show that the complex, crystallizing in the triclinic space group, undergoes a phase transition between 220 K and 230 K, connected with the doubling of cell volume. Crystal structures at 230 K (1a; HT phase) and 150 K (1b; LT phase) are described and a transition mechanism is discussed. In both phases the compound has an extended chain structure, in which the neutral molecule of N,N′-diethylnicotinamide acts as a bridging ligand binding through pyridine N atom to one centre and through amide O atom to the neighbouring Fe centre. The Fe²⁺ ion has a slightly distorted trans-octahedral environment with FeO₂N₄ chromophore, and all Fe-O and Fe-N bonds in the typical for high-spin iron(II) compounds range. Variable-temperature magnetic susceptibility data in the temperature range 1.8-300 K show that iron(II) is high-spin S = 2(⁵T_2g) and as a result effects due to zero-field splitting are anticipated at low temperatures. The IR spectrum suggested the coordination of N,N′-diethylnicotinamide to the central atom of iron(II) as a bridging ligand and NCS group as a monodentate ligand.     

Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of Ca(V)1.2

Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca²⁺ channel (Ca_V1.2) regulates Ca²⁺ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with Ca_V1.2 under low resting [Ca²⁺], but is poised to change conformation and position when intracellular [Ca²⁺] rises. CaM binding Ca²⁺, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A₁₅₈₈, and C₁₆₁₄ and the IQ motif studied as overlapping peptides IQ₁₆₄₄ and IQ′₁₆₅₀ as well as their effect on calcium binding. (Ca²⁺)₄-CaM bound to all four peptides very favorably (K_d ≤ 2 nM). Linkage analysis showed that IQ_1644-1670 bound with a K_d ~ 1 pM. In the pre-IQ region, (Ca²⁺)₂-N-domain bound preferentially to A₁₅₈₈, while (Ca²⁺)₂-C-domain preferred C₁₆₁₄. When bound to C₁₆₁₄, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM.