Solution Structure Of Ca2 Research Articles

NMR structure note: solution structure of Ca2+ binding domain 2B of the third isoform of the Na+/Ca2+ exchanger

NMR structure note: solution structure of Ca2+ binding domain 2B of the third isoform of the Na+/Ca2+ exchanger

Solution Structures of Ca2+-CIB1 and Mg2+-CIB1 and Their Interactions with the Platelet Integrin αIIb Cytoplasmic Domain

The calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous Ca(2+)-binding protein and a specific binding partner for the platelet integrin αIIb cytoplasmic domain, which confers the key role of CIB1 in hemostasis. CIB1 is also known to be involved in apoptosis, embryogenesis, and the DNA damage response. In this study, the solution structures of both Ca(2+)-CIB1 and Mg(2+)-CIB1 were determined using solution-state NMR spectroscopy. The methyl groups of Ile, Leu, and Val were selectively protonated to compensate for the loss of protons due to deuteration. The solution structure of Ca(2+)-CIB1 possesses smaller opened EF-hands in its C-domain compared with available crystal structures. Ca(2+)-CIB1 and Mg(2+)-CIB1 have similar structures, but the N-lobe of Mg(2+)-CIB1 is slightly more opened than that of Ca(2+)-CIB1. Additional NMR experiments, such as chemical shift perturbation and methyl group solvent accessibility as measured by a nitroxide surface probe, were carried out to further characterize the structures of Ca(2+)-CIB1 and Mg(2+)-CIB1 as well as their interactions with the integrin αIIb cytoplasmic domain. NMR measurements of backbone amide proton slow motion (microsecond to millisecond) dynamics confirmed that the C-terminal helix of Ca(2+)-CIB1 is displaced upon αIIb binding. The EF-hand III of both Ca(2+)-CIB1 and Mg(2+)-CIB1 was identified to be directly involved in the interaction of CIB1 with αIIb. Together, these data illustrate that CIB1 behaves quite differently from related EF-hand regulatory calcium-binding proteins, such as calmodulin or neuronal calcium sensor proteins.

Solution structure of the regulatory domain of human cardiac troponin C in complex with the switch region of cardiac troponin I and W7: The basis of W7 as an inhibitor of cardiac muscle contraction

The solution structure of Ca2+-bound regulatory domain of cardiac troponin C (cNTnC) in complex with the switch region of troponin I (cTnI147–163) and the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfinamide (W7), has been determined by NMR spectroscopy. The structure reveals that the W7 naphthalene ring interacts with the terminal methyl groups of M47, M60, and M81 as well as aliphatic and aromatic side chains of several other residues in the hydrophobic pocket of cNTnC. The H3 ring proton of W7 also contacts the methyl groups of I148 and M153 of cTnI147–163. The N-(6-aminohexyl) tail interacts primarily with the methyl groups of V64 and M81, which are located on the C- and D-helices of cNTnC. Compared to the structure of the cNTnC•Ca2+•W7 complex (Hoffman, R. M. B. and Sykes, B. D. (2009) Biochemistry 48, 5541–5552), the tail of W7 reorients slightly toward the surface of cNTnC while the ring remains in the hydrophobic pocket. The positively charged –NH3+ group from the tail of W7 repels the positively charged R147 of cTnI147–163. As a result, the N-terminus of the peptide moves away from cNTnC and the helical content of cTnI147–163 is diminished, when compared to the structure of cNTnC•Ca2+•cTnI147–163 (Li, M. X., Spyracopoulos, L., and Sykes B. D. (1999) Biochemistry 38, 8289–8298). Thus the ternary structure cNTnC•Ca2+•W7•cTnI147–163 reported in this study offers an explanation for the ∼13-fold affinity reduction of cTnI147–163 for cNTnC•Ca2+ in the presence of W7 and provides a structural basis for the inhibitory effect of W7 in cardiac muscle contraction. This generates molecular insight into structural features that are useful for the design of cTnC-specific Ca2+-desensitizing drugs.

Leucine 85 Is an Important Determinant of Divalent Ion Affinity in Rat β-Parvalbumin (Oncomodulin)

Despite 69% sequence identity with chicken parvalbumin 3 (CPV3), rat beta-parvalbumin (beta-PV) exhibits a substantially lower Ca(2+) affinity (DeltaDeltaG degrees ' = 2.0 kcal/mol). This difference largely reflects the disparate behavior of the respective CD sites. Replacement of the rat beta-PV codon with the CPV3 codon at positions 49, 50, and 57-60 produces virtual sequence identity with the CPV3 CD site. However, the resulting protein exhibits a modest (0.5 kcal/mol) improvement in Ca(2+) affinity, implying that sequence differences beyond the binding site modulate divalent ion binding behavior. The solution structure of Ca(2+)-free rat beta-PV suggested that Leu-85, phenylalanine in CPV3, might be an important determinant. Therefore, the impact of the L85F mutation on divalent ion affinity was examined in rat beta-PV, in the variant harboring all six of the aforementioned CD site mutations, and in the intermediate CD site variants. We find that the identity of residue 85, located within the E helix, strongly influences divalent ion affinity in the mammalian beta-PV isoform and that its impact is mediated by interactions with residues in the CD site. In the wild-type protein, L85F primarily affects the EF site. By contrast, in the presence of the six CD site mutations, L85F also improves the CD site performance, yielding a protein with Ca(2+) affinity rivaling that of CPV3 and markedly enhanced Mg(2+) affinity as well. The impact of L85F on CD site Ca(2+) affinity is particularly sensitive to the identities of residues 59 and 60. Interestingly, however, significant improvement in CD site Mg(2+) affinity also requires mutation of additional CD site residues.

Solution structure of Ca2+/calmodulin complexed with a lentivirus lytic peptide 1 reveals a novel mode of molecular recognition

Small-angle X-ray scattering was used to analyze the interaction of Ca2+/calmodulin (CaM) with a lentivirus lytic peptide 1 (LLP1) derived from the cytoplasmic tail of HIV-1 transmembrane glycoprotein. The synthetic peptide homologues of LLP1 were selected from three species of the glycoprotein: ENV_HV1A2, ENV_HV1B1 and ENV_HV1H2. Ca2+/CaM binds LLP1 with the truncation of three or ten residues and adopts almost the same globular structure as that of the complex with a peptide from myosin light chain kinase (MLCK), indicating that the Ca2+/CaM-binding site locates on the shorter sequence. Moreover, Ca2+/CaM binds a peptide with the opposite sequence and adopts almost the same globular structure as that in the original sequence. Taken together, the results provide evidence that LLP1 can bind to the N- and C-terminal lobes of CaM with a polarity opposite to that observed for the CaM–MLCK complex and the binding mode of Ca2+/CaM molecular recognition is well preserved despite the sequence variation in the three species, suggesting that this region of the transmembrane glycoprotein is important to viral replication.

Solution structure of Ca(2+)-calmodulin reveals flexible hand-like properties of its domains.

The solution structure of Ca(2+)-ligated calmodulin is determined from residual dipolar couplings measured in a liquid crystalline medium and from a large number of heteronuclear J couplings for defining side chains. Although the C-terminal domain solution structure is similar to the X-ray crystal structure, the EF hands of the N-terminal domain are considerably less open. The substantial differences in interhelical angles correspond to negligible changes in short interproton distances and, therefore, cannot be identified by comparison of NOEs and X-ray data. NOE analysis, however, excludes a two-state equilibrium in which the closed apo conformation is partially populated in the Ca(2+)-ligated state. The difference between the crystal and solution structures of Ca(2+)-calmodulin indicates considerable backbone plasticity within the domains of calmodulin, which is key to their ability to bind a wide range of targets. In contrast, the vast majority of side chains making up the target binding surface are locked into the same chi(1) rotameric states as in complexes with target peptide.

Calcium-induced conformational transition revealed by the solution structure of apo calmodulin

The solution structure of Ca(2+)-free calmodulin has been determined by NMR spectroscopy, and is compared to the previously reported structure of the Ca(2+)-saturated form. The removal of Ca2+ causes the interhelical angles of four EF-hand motifs to increase by 36 degrees-44 degrees. This leads to major changes in surface properties, including the closure of the deep hydrophobic cavity essential for target protein recognition. Concerted movements of helices A and D with respect to B and C, and of helices E and H with respect to F and G are likely responsible for the cooperative Ca(2+)-binding property observed between two adjacent EF-hand sites in the amino- and carboxy-terminal domains.

Solution structure of calmodulin and its complex with a myosin light chain kinase fragment

The solution structure of Ca 2+ ligated calmodulin and of its complex with a 26-residue peptide fragment of skeletal muscle myosin light chain kinase (skMLCK) have been investigated by multi-dimensional NMR. In the absence of peptide, the two giobular domains of calmodulin adopt the same structure as observed in the crystalline form [2]. The so-called ‘central helix’ which is observed in the crystalline state is disrupted in solution. 15N relaxation studies show that residues Asp78 through Ser81, located near the middle of this ‘central helix’, form a very flexible link between the two globular domains. In the presence of skMLCK target peptide, the peptide-protein complex adopts a globular ellipsoidal shape. The helical peptide is located in a hydrophobic channel that goes through the center of the complex and makes an angle of ∼45° with the long axis of the ellipsoid.