Valerie B. Patchell

Binding sites involved in the interaction of actin with the N-terminal region of dystrophin

Two actin‐binding sites have been identified on human dystrophin by proton NMR spectroscopy of synthetic peptides corresponding to defined regions of the polypeptide sequence. These are Actin‐Binding Site 1 (ABS1) located at residues 17–26 and Actin‐Binding Site 2 (ABS2) in the region of residues 128–156. Using defined fragments of the actin amino acid sequence, ABS1 has been shown to bind to actin in the region represented by residues 83–117 and ABS2 to the C‐terminal region represented by residues 350–375. These dystrophin‐binding sites lie on the exposed domain in the actin filament.

The interaction of actin with dystrophin.

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.

Sequential phosphorylation of adjacent serine residues on the N-terminal region of cardiac troponin-I: structure-activity implications of ordered phosphorylation

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.

Two-site phosphorylation of the phosphorylatable light chain (20-kDa light chain) of chicken gizzard myosin

When prepared under specified conditions chicken gizzard myosin was obtained which when incubated with ATP gave rise to a diphosphorylated as well as the monophosphorylated form of P light chain. Formation of the diphosphorylated light chain occurred more readily with these myosin preparations, but could also be obtained by prolonged incubation of the isolated whole light chain fraction with kinase preparations from rabbit skeletal and chicken gizzard muscles. Using isolated light chains as substrate the more readily formed monophosphorylated light chain contained serine phosphate while the diphosphorylated form contained serine and threonine phosphates.

Phosphorylation of the minimal inhibitory region at the C-terminus of caldesmon alters its structural and actin binding properties

Caldesmon is an inhibitory protein believed to be involved in the regulation of thin filament activity in smooth muscles and is a major cytoplasmic substrate for MAP kinase. NMR spectroscopy shows that the actin binding properties of the minimal inhibitory region of caldesmon, residues 750-779, alter upon MAP kinase phosphorylation of Ser-759, a residue not involved in actin binding. This phosphorylation leads to markedly diminished actin affinity as a result of the loss of interaction at one of the two sites that bind to F-actin. The structural basis for the altered interaction is identified from the observation that phosphorylation destabilises a turn segment linking the two actin binding sites and thereby results in the randomisation of their relative disposition. This modulatory influence of Ser-759 phosphorylation is not merely a function of the bulkiness of the covalent modification since the stability of the turn region is observed to be sensitive to the ionisation state of the phosphate group. The data are discussed in the context of the inhibitory association of the C-terminal domain of caldesmon with F-actin.

The Regulatory Effects of Tropomyosin and Troponin-I on the Interaction of Myosin Loop Regions with F-actin

The N terminus of skeletal myosin light chain 1 and the cardiomyopathy loop of human cardiac myosin have been shown previously to bind to actin in the presence and absence of tropomyosin (Patchell, V. B., Gallon, C. E., Hodgkin, M. A., Fattoum, A., Perry, S. V., and Levine, B. A. (2002) Eur. J. Biochem. 269, 5088–5100). We have extended this work and have shown that segments corresponding to other regions of human cardiac β-myosin, presumed to be sites of interaction with F-actin (residues 554–584, 622–646, and 633–660), likewise bind independently to actin under similar conditions. The binding to F-actin of a peptide spanning the minimal inhibitory segment of human cardiac troponin I (residues 134–147) resulted in the dissociation from F-actin of all the myosin peptides bound to it either individually or in combination. Troponin C neutralized the effect of the inhibitory peptide on the binding of the myosin peptides to F-actin. We conclude that the binding of the inhibitory region of troponin I to actin, which occurs during relaxation in muscle when the calcium concentration is low, imposes conformational changes that are propagated to different locations on the surface of actin. We suggest that the role of tropomyosin is to facilitate the transmission of structural changes along the F-actin filament so that the monomers within a structural unit are able to interact with myosin.

Importance of the C-terminus of the human 5-HT3A receptor subunit.

Amongst the family members of Cys-loop LGICs, the atypical ability of the 5-HT3A subunit to form functional homomeric receptors allowed a direct investigation of the role of the C-terminus. Deletion of the three C-terminal amino acids (DeltaGln453-DeltaTyr454-DeltaAla455) from the h5-HT3A subunit prevented formation of a specific radioligand binding site as well as expression within the cell membrane. Removal of merely the C-terminal residue (DeltaAla455) reduced specific radioligand binding (to 4+/-1% relative to the wild-type; cells grown at 37 degrees C) and also cell membrane expression; these reductions were less evident when the DeltaAla455 expressing cells were grown at 27 degrees C (specific radioligand binding levels 27+/-5% relative to wild-type also grown at 27 degrees C). Mutation of the h5-HT3A C-terminal amino acid, alanine, for either glycine (Ala455Gly), valine (Ala455Val) or leucine (Ala455Leu) reduced specific radioligand binding levels by 24+/-23%, 32+/-12% and 88+/-1%, respectively; the latter mutant also displaying reduced membrane expression. In contrast, mutation to alanine of the two amino acids preceding the C-terminal alanine (Gln453Ala and Tyr454Ala) had no detrimental effects on specific radioligand binding or cell membrane expression levels. The present study demonstrates an important role for the C-terminus in the formation of the functional h5-HT3A receptor. The partial restoration of 5-HT3 receptor binding and cell membrane expression when cells expressing C-terminal mutant 5-HT3A subunits were grown at a lower temperature (27 degrees C) suggests that the C-terminus stabilises the 5-HT3 receptor allowing subunit folding and subsequent maturation.

Phosphorylation of chicken gizzard myosin and the Ca2+-sensitivity of the actin-activated Mg2+-ATPase

A method is described for the preparation of partially and fully phosphorylated chicken gizzard myosin. When fully phosphorylated it possessed an actin‐activated Mg2+‐ATPase of similar specific activity to that of mammalian skeletal muscle myosin. The Mg2+‐ATPase activity of these preparations was related in a non‐linear fashion to increasing phosphorylation of the P light chain. When P light chain phosphorylation occurred during enzymic assay the Mg2+‐ATPase activity remained constant. Fully phosphorylated preparations of gizzard myosin possessed an actin‐activated Mg2+‐ATPase that was not Ca2+‐sensitive, whereas the Mg2+‐ATPase of partially phosphorylated myosin preparations was Ca2+‐sensitive.

Use of Synthetic Peptides in the Study of the function of Dystrophin

From the work of the Kunkel (Koenig et al., 1987) and Worton groups (Burgess et al., 1987) it has been established that the protein dystrophin is absent or in some cases present in a modified form in patients with Duchenne and Becker muscular dystrophies (Xp21 myopathies). The amino acid sequence deduced from analysis of the complementary DNA indicates that dystrophin from human skeletal muscle has a molecular weight of 427,000 and consists of a polypeptide chain of 3,865 residues with a multi-domain structure (Koenig et al., 1988). The N-terminal domain of 240 residues shows homology to the N-terminal domain of the α-actinins in which region actin has been shown to bind (Mimura & Asano, 1986). The second and largest domain is formed by a succession of 25 triple helical segments (the fourth of which is truncated) with loose homology between them and which show weak similarity to similar repeats in a and β-spectrin. A third domain extending over 150 residues is cysteine-rich and is similar in sequence to the carboxy terminal domain of α-actinin that contains two calcium binding sites. The corresponding sites on dystrophin are rather rudimentary and may not be functional.