Calmodulin myosin light chain kinase binding

Smooth muscle contractions are subject to the actions of hormones and related agents. As shown in Figure 17.32, binding of the hormone epinephrine to smooth muscle receptors activates an intracellular adenylyl cyclase reaction that produces cyclic AMP (cAMP). The cAMP serves to activate a protein kinase that phosphorylates the myosin light chain kinase. The phosphorylated MLCK has a lower affinity for the Ca -calmodulin complex and thus is physiologically inactive. Reversal of this inactivation occurs via myosin light chain kinase phosphatase. 

Smooth muscle sarcoplasm contains a myosin light chain kinase that is calcium-dependent. The Ca activation of myosin fight chain kinase requires binding of calmodulin-4Ca to its kinase subunit (Figure 49-14). 

Figure 11.9 (Left) The EF hand helix-loop-helix motif (centre) rat testes calmodulin. The globular domains each have two Ca2+-binding sites, indicated by white spheres, connected by a seven-turn a-helix (right) two views of the (Ca2+)4 fruit fly calmodulin in complex with its 26-residue target peptide from rabbit skeletal muscle myosin light chain kinase, ((left, centre) From Voet and Voet, 2004. Reproduced with permission from John Wiley Sons., Inc. and (right) Carafoli, 2002. Copyright (2002) National Academy of Sciences, USA.)...

The calcium mediated contraction of smooth muscle, which unlike striated muscle does not contain troponin, is quite different and requires a particular calcium-binding protein called calmodulin. Calmodulin (CM) is a widely distributed regulatory protein able to bind, with high affinity, four Ca2+ per protein molecule. The calcium—calmodulin (CaCM) complex associates with, and activates, regulatory proteins, usually enzymes, in many different cell types in smooth muscle the target regulatory proteins are caldesmon (CDM) and the enzyme myosin light chain kinase (MLCK). As described below, CaCM impacts on both actin and myosin filaments. 

Figure 22.12 Regulation of actin-myosin interaction in smooth muscle via the light-chain kinase and phosphatase and effect on blood pressure. ions bind to calmodulin and the complex stimulates the conversion of inactive myosin light chain kinase (MLCK) to active MLCK which then phosphorylates the light chain. This results in activation of the cross-bridge cycle. The overall effect is vasoconstriction of the arteriole, which increases blood pressure.

Fig. 6.10. Structure of Ca -cal-modulin. a) Structure of free Ca -calmodulin. Calmodulin is composed of two domains that can each bind two Ca ions. Both domains are linked via a flexible structural element. The Ca ions are represented as spheres. MOLSKRIPT representation according to Krauhs, (1991). b) Ca -calmodulin in complex with a peptide that is derived from the Ca -binding domain of the myosin light chain kinase. Ca -calmodulin is represented as a band, and the peptide substrate is a ball and stick" model. The perspective is along the longitudinal axis of the hnear peptide substrate.

Hathaway, D.R. Adelstein, R.S. Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity. Proc. Natl. Acad. Sci. USA, 76, 1653-1657 (1979)... 

Toeroek, K. Cowley, D.J. Brandmeier, B.D. Howell, S. Aitken, A. Tren-tham, D.R. Inhibition of calmodulin-activated smooth-muscle myosin light-chain kinase by calmodulin-binding peptides and fluorescent (phosphodiesterase-activating) calmodulin derivatives. Biochemistry, 37, 6188-6198 (1998)... 

Padre, R.C. Stull, J.T. Conformational requirements for Ca /calmodulin binding and activation of myosin light chain kinase. FEES Lett., 472, 148-152 (2000)... 

Fig. 9. Helical net diagram (Crick, 1953) of a model calmodulin-binding peptide and the putative calmodulin-binding domains of two forms of myosin light-chain kinase (MLCK). The sequences are drawn together on a single helical net and are taken from (clockwise from left) the model peptide described by DeGrado et al. (1985), skeletal muscle MLCK peptide 342-359 (Edelman et al., 1985), and the N-terminal 18 residues of a peptide derived from smooth muscle MLCK (Lucas et al., 1986). The amino acids in the sequences are given in single letter codes. Positions that are hydrophobic in all three sequences are indicated by shading.

In smooth muscle, P-adrenoceptors decrease contractility PKA phosphorylates MLCK, which thereby becomes inactivated. In contrast, aj-adrenoceptors increase smooth muscle contractility. They activate phospholipase C, which in turn releases inositoltriphosphate (IP3) from the endoplasmic reticulum by binding to a cognate receptor channel (Figure 6.10c). Ca then binds to calmodulin, which in turn activates myosin light chain kinase. 

Figure 6.10. Calcium-dependent signalling by adrenergic receptors. a p-Adrenergic receptors activate adenylate cyclase. cAMP activates protein kinase A (PKA). In heart muscle, PKA phospho-rylates several Ca transporters and charmels, so that the amount of Ca available for contraction is increased. PL Phospholam-ban SERCA SR/ER Ca transporter, b In smooth muscle, myosin activation in works by way of phosphorylation, which is performed by myosin light chain kinase (MLCK). Inactivation is accomplished by myosin light chain phosphatase (MLCP). c aj-Adrenergic receptors stimulate phospholipase C, which releases inositoltriphosphate (IP3). IP3 binds to a cognate ligand-gated Ca chaimel in the ER and releases Ca, which with calmodulin activates MLCK.

Figure 2. Improvement of yellow cameleon by using a circularly permuted YFP variant. (A) The three-dimensional structure of GFP with the positions of the original (Metl) and new N-termini (Aspl73). (B) Domain structures of YCS.12 and YC3.60. CaM, Xenopus calmodulin Ml3, a CaM binding peptide derived from myosin light chain kinase (C, D) Emission spectra of YCS. 12 (C) and YC3.60 (D) (excitation at 435 nm) at zero (doted line) and saturated Ca (solid line). (E) A series of confocal pseudo-B/W images showing propagation of [Ca c- These images were taken at...

The value of X (Eqn. 2) appears to be variable depending on the number of binding subunits for a given enzyme. For example, the cyclic nucleotide phosphodiesterase is frequently described as being a dimeric molecule with each of the two monomers capable of binding one molecule of calmodulin. Similarly, the myosin light chain kinase is reported to bind one calmodulin molecule per subunit (51). Multiple calmodulin binding sites per monomer have not as yet been described for any enzyme with the possible exception of phosphorylase Id kinase. 

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