Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase.

110

100

The mechanism of the interactions of 2-chloro-(epsilon-amino-Lys75)-[6-[4-(N,N-diethylamino)phenyl]- 1,3,5-triazin-4-yl]calmodulin (TA-calmodulin) with smooth muscle myosin light-chain kinase (MLCK) and two 17-residue peptides, Ac-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-CONH2 (Trp peptide) and Tyr peptide, in which W is replaced by Y, were studied by measurements of equilibrium and transient fluorescence changes in the nanomolar range. Most reactions were carried out in 100 microM CaCl2 at ionic strength 0.15 M, pH 7.0, and 21 degrees C. In each case association of MLCK or peptide to TA-calmodulin could be described by a two-step process, a bimolecular step and an isomerization. In the case of the interaction between TA-calmodulin and Tyr peptide it was shown that the isomerization involved the binary complex of TA-calmodulin and Tyr peptide as opposed to an isomerization of either TA-calmodulin or Tyr peptide in isolation. These distinctions depended in part on development for transient kinetic experiments of a general theory to quantify relative phase amplitudes in two-step mechanisms. The kinetics for all three association reactions were then interpreted in terms of a bimolecular association (rate constants k+1 and k-1) followed by an isomerization of the binary complex (rate constants k+2 and k-2). For the interaction of TA-calmodulin and Tyr peptide, values of the rate constants are k+1, 8.8 x 10(8) M-1 s-1; k-1, 5.7 s-1; k+2, 0.38 s-1; and k-2, 0.65 s-1. The fluorescence intensities (lambda ex 365 nm, lambda ex 365 nm, lambda em > 400 nm) of TA-calmodulin, the initial binary complex of TA-calmodulin and Tyr peptide, and the isomerized binary complex are in the ratio 1:2.8:1.3. Analogous mechanisms were found for TA-calmodulin binding to Trp peptide and to MLCK, but values for the rate constants and relative fluorescence intensities of the binary complexes were generally not so completely defined. Values for the Trp peptide and MLCK, respectively, are k+1, 8.8 x 10(8) M-1 s-1 and 1.1 x 10(8) M-1 s-1; (k+2 + k-2), 0.97 s-1 and 1.3 s-1; and k-1k-2/(k+2 + k-2), 0.0079 s-1 and 0.025-0.056 s-1. Equilibrium dissociation constants (Kd) for interactions of TA-calmodulin and targets determined from these data are Tyr peptide, 4.1 nM; Trp peptide, 0.011 nM; and MLCK, 0.23-0.51 nM.(ABSTRACT TRUNCATED AT 400 WORDS)

Section: Discussionmentioning

confidence: 62%

Mechanism of2-Chloro-(.epsilon.-amino-Lys75)-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin Interactions with Smooth Muscle Myosin Light Chain Kinase and Derived Peptides

Török¹,

Trentham²

1994

110

100

“…The pattern of residues for the binding of the amino-terminal domain of CaM is less conserved on MyoD. in particular, two hydrophobic residues (residues 9 and 18) that may stabilize the CaM-target complexes (Ikura et al, 1992;Bagchi et al, 1992) are changed to hydrophilic residues in the MyoD proteins. This may lead to a reduced affinity between MyoD and CaM compared to CaM-target enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…1992). The a-helix I motif in myogenic proteins has in common with the CaM-binding domain of CaM-target enzymes the presence of aromatic or aliphatic residues (white boxes) critical for hydrophobic interaction with the carboxyl-terminal domain of CaM and for high-affinity interactions (Bagchi et al. 1992;Ikura et al.…”

Section: Methodsmentioning

confidence: 99%

“…1992). The carboxy-terminus of the CaM binding domain on CaM-target enzymes also has an arginine residue that was found to be critical for CaM binding (Bagchi et al, 1992); this residue is also present in the putative CaM binding domain of myogenic proteins. Note also the presence of a cluster of basic amino acids (arrows) present in all CaM-target peptides and myogenic proteins.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Interactions of Myogenic bHLH Transcription Factors with Calcium-Binding Calmodulin and S100a (.alpha..alpha.) Proteins

Baudier

Bergeret²,

Bertacchi³

et al. 1995

MyoD belongs to a family of myogenic basic helix-loop-helix (bHLH) transcription factors that activate muscle-specific genes. The basic helix I sequence of the bHLH motif contains a consensus sequence for protein kinase C (PKC) substrates. We show here that MyoD is indeed phosphorylated by PKC in vitro on Thr 115 within the basic part of the bHLH motif. By analogy with calmodulin-target peptide models, we also identified within the consensus basic helix I motif of myogenic proteins a conserved putative calmodulin/S100-binding domain. Calcium-dependent interaction between MyoD with calmodulin and the abundant muscle S100a(alpha alpha) proteins was demonstrated by affinity chromatography and cross-linking experiments. The binding of calmodulin and S100a inhibited MyoD phosphorylation by PKC as well as MyoD DNA binding activity. S100a was found to be more efficient than calmodulin in antagonizing DNA binding to MyoD. We next developed a rapid purification method for bacterial recombinant MyoD-bHLH domain by affinity chromatography using a calmodulin-Sepharose column and investigated the phosphorylation of that peptide by PKC and its interactions with calmodulin and S100a. We confirmed the phosphorylation of the threonine residue 115 in the MyoD-bHLH by PKC with a Km of 0.8 microM. Calmodulin and S100a binding inhibited MyoD-bHLH phosphorylation by PKC. A strict calcium-dependent interaction between calcium binding proteins and the MyoD-bHLH was identified by native gel electrophoresis and fluorescence spectroscopy with 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin. The MyoD-bHLH bound to fluorescently labeled 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin with a dissociation constant around 20 nM. S100a inhibited stoichiometrically the binding of the bHLH peptide for labeled calmodulin, suggesting an affinity of S100a for the bHLH peptide at least 1 order of magnitude higher than calmodulin. In favor of an in vivo interaction between S100a and MyoD, we report that S100a- and MyoD-like immunoreactivities colocalize in H9c2 cells, and that a significant amount of MyoD-like immunoreactivity is recovered in the S100a immunoprecipitate from crude H9c2 cell extract in the presence of calcium. We propose that myogenic proteins represent a new family of calmodulin/S100-binding PKC substrates and that calmodulin/S100a could participate in the regulation of the bHLH myogenic protein activities.

“…The possibility that enzyme-derived CBPs, represented in this study by M5 and C28W, may also be involved in certain discrete interactions of their respective enzymes with membrane lipids can be postulated, on the basis of the results presented above and in the previously mentioned work on the stimulation of the plasma membrane Ca2+ pump with acidic phospholipids (Brodin etal., 1992). Moreover, these peptides have the capacity to assume an a-helical conformation (Garone & Steiner, 1990;Toma et al, 1981;Vorherr et al, 1990) which appears to be mandatory for establishing the hydrophobic interaction with CaM (Bagchi et al, 1992) and with the endogenous autoinhibitory pseudosubstrate sites in the respective CaM-dependent enzymes (Kennedy et al, 1987;Vorherr etal., 1990). The occurrence of amphipathic a-helixforming peptides in amphitropic enzymes is recently being recognized as potentially important in their interaction with and translocation to membranes.…”

Section: Discussionmentioning

confidence: 99%

Synthetic peptides corresponding to the calmodulin-binding domains of skeletal muscle myosin light chain kinase and human erythrocyte calcium pump interact with and permeabilize liposomes and cell membranes

Eshel

Shai²,

Vorherr³

et al. 1993

Synthetic calmodulin-binding (CaM-binding) peptides (CBPs) representing CaM-binding domains of Ca2+/CaM-dependent enzymes have been reported to interfere with the activity of the melanocyte-stimulating hormone (MSH) receptor function in melanoma cells [Gerst, J. E., & Salomon, Y. (1988) J. Biol. Chem. 263, 7073-7078]. We postulated that membrane lipids may play an important role in the mode of action of CBPs on cells. We therefore tested the ability of CBPs to interact with membrane bilayers. Using artificial phospholipid vesicles, or M2R melanoma cells and cell membranes derived therefrom, as models, we report here that synthetic peptides representing the CaM-binding domains of skeletal muscle myosin light chain kinase (M5) and the human erythrocyte calcium pump (C28W), as well as other CBPs, interact with lipid bilayers and cell membranes. Significant interactions of CBPs with the lipid bilayer were detected in both model systems. M5 and C28W were found to partition into the lipid bilayer of melanoma cell membranes and soybean lecithin vesicles, and surface partition constants obtained (for the liposome model) were in the range 10(3)-10(4) M-1. In addition, C28W and its N-modified NBD derivative were found to inhibit [125I]iodo-[Nle4,D-Phe7]alpha MSH binding to cultured M2R melanoma cells. These and other CBPs were also found to induce the release of cations and calcein from liposomes, suggesting that the interaction of CBPs with the lipid bilayer increases membrane permeability.(ABSTRACT TRUNCATED AT 250 WORDS)