Two actinmbinding sites have been identified on human dystrophin by proton NMR spectroscopy of synthetic peptides corresponding to delined regions of the polypeptidcsequencc. These are Act&Binding Site I (ABSI ) located at residues 17-26 and Actin-Binding Site 2 (AEtS2) in the region oi residues 125-156. Using defined fragments of the actin amino acid sequence. ABSI has been shown to bind to actin in the region represented by residues 83-1 I7 and ABS2 to the C-terminal region represented by residues 350-375. These dystrophin-binding sites lie on the exposed domain in the uctin filament.

Proton NMR spectroscopy of synthetic peptides corresponding to defined regions of human dystrophin has been employed to study the interaction with F‐actin. No evidence of interaction with a C‐terminal region corresponding to amino acid residues 3429–3440 was obtained. F‐actin restricted the mobility of residues 19–27 in a synthetic peptide corresponding to residues 10–32. This suggests that this is a site of F‐actin interaction in the intact dystrophin molecule. Identical sequences to that of residues 19—22 in dystrophin, namely Lys‐Thr‐Phe‐Thr are also present in the N‐terminal regions of the α‐actinins implying this is also a site of F‐actin interaction with α‐actinin.

The interaction between actin and caldesmon that is associated with the inhibition of actomyosin ATPase activity in smooth muscle has been studied using 'H-NMR spectroscopy. Binding studies using the intact molecules were complemented by the use of thrombic cleavage fragments of both turkey and chicken gizzard caldesmon as well as defined peptides of actin, in order to investigate the conformational properties of caldesmon and to localise regions of the primary structures that participate in protein-protein contacts. The binding of caldesmon is shown to involve distinct segments on the N-terminal region (residues 1-44) of actin, as previously observed for the inhibitory component of the thin filament of striated muscle, troponin I [Levine et al. (1988) Eur. J . Biochem. 153, 389 -3971. The comparable structural properties of these tissue-specific inhibitors of actomyosin ATPase and the similarities in their mode of interaction at the N-terminal region of actin suggest common aspects to the structural mechanism for thin-filament regulation in smooth and striated muscle. Unlike the inhibitory interaction of troponin I, however, the binding of caldesmon to the N-terminal region of actin directly involves groups within residues 20 -41 of actin that are also recognised by myosin subfragment 1. The complementary segment of caldesmon has been localised to a 15-kDa thrombic fragment (residues 483 -578) derived from the N-terminal portion of a 35-kDa proteolytic cleavage product from the C-terminal of caldesmon whose interaction with actin is modulated by calmodulin. The results are discussed in relation to the calcium-mediated mechanism for thinfilament regulation in smooth and striated muscle.The contractile response of vertebrate smooth muscle requires the calcium-dependent phosphorylation of the P light chain of myosin [I, 21. A further regulatory mechanism in smooth muscle was subsequently identified and shown to involve the inhibition of actin-activated ATPase of phosphorylated smooth muscle myosin by caldesmon, a component of the thin filament in smooth muscle [2-61. In a manner analogous to the calcium-modulated inhibitory interaction between the troponin complex and actin on the thinfilament of skeletal muscle, inhibition by caldesmon can be mediated by its association with calmodulin in the presence of calcium [6 -91. As is also the case with the troponin complex, inhibition of actomyosin activity by caldesmon is enhanced by tropomyosin [5,8, 101. Caldesmon has further been shown to also bind to the tail (subfragment 2) region of myosin [ll -141 suggesting that, unlike troponin, it may form a calcium-dependent cross-link between actin and myosin in smooth muscle.Electron microscopic images [15] and sedimentation velocity experiments [16] indicate that chicken gizzard caldesmon (molecular mass = 87 kDa [17]) is an elongated molecule. It is often assumed therefore that caldesmon, like tropomyosin, spans the actin filament in order to enable its various interactions with the other components of the protein assemb...

We have used NMR spectroscopy to monitor the phosphorylation of a peptide corresponding to the N-terminal region of human cardiac troponin-I (residues 17-30), encompassing the two adjacent serine residues of the dual phosphorylation site. An ordered incorporation of phosphate catalysed by PKA was observed, with phosphorylation of Ser-24 preceding that of Ser-23. Diphosphorylation induced a conformational transition in this region, involving the specific association of the Arg-22 and Ser-24P side-chains, and maximally stabilised when both phosphoserines were in the di-anionic form. The results suggest that the second phosphorylation at Ser-23 of cardiac troponin-I is of particular significance in the mechanism by which adrenaline regulates the calcium sensitivity of the myofibrillar actomyosin Mg-ATPase.

Proton NMR studies have shown that when a peptide corresponding to the N-terminal region of phospholamban, PLB(1±20), interacts with the Ca 2+ ATPase of the sarcoplasmic reticulum, SERCA1a, docking involves the whole length of the peptide. Phosphorylation of Ser16 reduced the affinity of the peptide for the pump by predominantly affecting the interaction with the C-terminal residues of PLB(1±20). In the phosphorylated peptide weakened interaction occurs with residues at the N-terminus of PLB(1±20). PLB(1±20) is shown to interact with a peptide corresponding to residues 378±405 located in the cytoplasmic region of SERCA2a and related isoforms. This interaction involves the C-terminal regions of both peptides and corresponds to that affected by phosphorylation. The data provide direct structural evidence for complex formation involving residues 1±20 of PLB. They also suggest that phospholamban residues 1±20 straddle separate segments of the cytoplasmic domain of SERCA with the N-terminus of PLB associated with a region other than that corresponding to SERCA2a(378±405).Keywords: phospholamban; calcium-activated ATPase; sarcoplasmic reticulum; phosphorylation; NMR.The Ca 2+ ATPase of the sarcoplasmic reticulum (SERCA) is a calcium pump which functions in association with the Ca 2+ release channels, and is responsible for maintaining the calcium transients upon which the regulation of contractile activity in all types of muscle depends. The SERCA enzyme family consists of a number of isoforms encoded by three genes that are expressed in a tissue-specific manner. In cardiac muscle the isoform characteristic of that tissue, SERCA2a, is associated with phospholamban (PLB), a 52-residue polypeptide whose reversible phosphorylation enables the calcium pump to be modulated [1,2]. Interestingly, the same two proteins are expressed in slow-twitch skeletal muscle [3,4], whereas different isoforms, SERCA1a and 1b [5] but no PLB [6] are present in fast-twitch skeletal muscle. Nevertheless, SERCA isolated from fast skeletal muscle can be regulated by PLB in vitro [7±9], although it has been suggested that it is regulated in situ by a related protein, sarcolipin [10].The presence of PLB enables the calcium pumping rate of the sarcoplasmic reticulum to be increased in cardiac, and to a lesser extent in slow-twitch skeletal muscles, in response to b-adrenergic stimulation. Although the detailed mechanism of this process is not understood, a current view is that in the unphosphorylated form PLB interacts with SERCA and inhibits pumping activity, possibly by reducing its affinity for calcium ions (compare [11] with [12,13]), and reducing the maximum turnover rate at saturating calcium ion concentration, which is reflected in the Ca/MgATPase activity rate (reviewed in [14,15]). Phosphorylation of Ser16 of PLB by cAMP or cGMP dependent protein kinases and/or phosphorylation of Thr17 by the multifunctional calmodulin dependent protein kinase [16,17] results in increased activity of the pump [18,19].The detailed mechanism by which PLB m...