Biomedical subjects

J Kendrick-Jones

Publications and source records attributed to J Kendrick-Jones.

At least 19 recordsLinked to original sources

PubMed1992

Primary structure of dystrophin-related protein.

Dystrophin-related protein (DRP or 'utrophin') is localized in normal adult muscle primarily at the neuromuscular junction. In the absence of dystrophin in Duchenne muscular dystrophy (DMD) patients, DRP is also present in the sarcolemma. DRP is expressed in fetal and regenerating muscle and may play a similar role to dystrophin in early development, although it remains to be determined whether DRP can functionally replace dystrophin in adult tissue. Previously we described a 3.5-kilobase complementary DNA clone that exhibits 80 per cent homology to the C-terminal domain of dystrophin. This sequence identifies a 13-kilobase transcript that maps to human chromosome 6 (refs 2, 11). Antibodies raised against the gene product identify a polypeptide with a relative molecular mass of about 400K in all tissues examined. To investigate the relationship between DRP and dystrophin in more detail, we have cloned and sequenced the whole DRP cDNA. Homology between DRP and dystrophin extends over their entire length, suggesting that they derive from a common ancestral gene. Comparative analysis of primary sequences highlights regions of functional importance, including those that may mediate the localization of DRP and dystrophin in the muscle cell.

Actinin↗

PubMed1992

Uncoupling of actin-activated myosin ATPase activity from actin binding by a monoclonal antibody directed against the N-terminus of myosin light chain 1.

The role of the N-terminal region of myosin light chain 1 (LC1) in actomyosin interaction was investigated using an IgG monoclonal antibody (2H2) directed against the N-terminal region of LC1. We defined the binding site of 2H2 by examining its cross-reactivity with myosin light chains from a variety of species and with synthetic oligopeptides. Our findings suggest that 2H2 is directed against the N-terminal region of LC1 which includes the trimethylated alanine residue at the N-terminus. In the presence of 2H2, the rate of actomyosin superprecipitation was reduced, although the extent was not. 2H2 caused a reduction in the Vmax of both myosin and chymotryptic S1(A1) actin-activated ATPase activity, while the Km appeared to be unaltered. The Mg(2+)-ATPase activity of myosin alone was also unaffected. Binding studies revealed that 2H2 did not prevent the formation of acto-S1 complex, either in the presence or in the absence of ATP, nor did it affect the ability of ATP to dissociate S1 from F-actin. Our findings suggest that the N-terminal region of LC1 is not essential for actin binding but is involved in modulating actin-activated ATPase activity of myosin.

Actins↗

PubMed1992

Expression of the N-terminal domain of dystrophin in E. coli and demonstration of binding to F-actin.

The N-terminal head domain of human dystrophin has been expressed in soluble form and high yield in E. coli, allowing us to test the previously unconfirmed assumption that dystrophin binds actin. DMD246, the first 246 amino acid residues of dystrophin, binds F-actin in a strongly co-operative manner with a Hill constant of 3.5, but does not bind G-actin. Dystrophin heads are thus functionally competent actin-binding proteins. This result opens the way to identifying critical residues in the actin-binding site and encourages us that the other domains of dystrophin might also be treated as functionally autonomous modules, accessible to a similar approach.

Actins↗

PubMed1992

Hybrid myosin light chains containing a calcium-specific site from troponin C.

Recombinant DNA approaches have allowed us to probe the mechanisms by which the regulatory light chains (RLCs) regulate myosin function by identifying the functional importance of specific regions of the RLC molecule. For example, we have demonstrated that the presence of high-affinity Ca2+/Mg(2+)-binding site in the N-terminal domain of the RLC is essential for the regulation of myosin-actin interaction [Reinach, F. C., Nagai, K. & Kendrick-Jones, J. (1986) Nature 322, 80-83]. To explore further the role of this metal-binding site in the RLC and generate an RLC with a Ca(2+)-specific site, we constructed four chicken skeletal muscle myosin regulatory light chain hybrid 'genes'. In these, the first domain containing the high-affinity Ca2+/Mg(2+)-binding site in the RLC was replaced with that containing the lower-affinity, Ca(2+)-specific, regulatory site from troponin C (TnC). In two of these hybrids, we replaced only the Ca(2+)-binding EF hand, while in the other two the EF hand and the N-terminal helix of TnC were transplanted. These hybrids were expressed in Escherichia coli in high yields and the purified proteins were used in calcium-binding experiments to assay the affinity and specificity of the sites and incorporated into scallop myosin to assay their regulatory behaviour. The results obtained show that the calcium-binding site from TnC, when transplanted into the RLC backbone, had a low affinity although most of its specificity appeared to be retained. As a result, although the TnC/RLC hybrids bound to scallop myosin and were able to activate the MgATPase activity of scallop acto-myosin, they were unable to regulate it. These results are in agreement with our previous findings that occupancy of the Ca2+/Mg2+ site in the RLC is essential for regulation. Our results suggest that the specificity and affinity of the calcium-binding site in troponin C is dependent on both intra- and inter-domain interactions within troponin C and that these latter interactions appear to be missing when this binding site is transplanted into the light chain backbone.

Amino Acid Sequence↗

PubMed1992

Chimeric myosin regulatory light chains identify the subdomain responsible for regulatory function.

Regulatory light chains, located on the 'motor' head domains of myosin, belong to the family of Ca2+ binding proteins that consist of four 'EF-hand' subdomains. Vertebrate regulatory light chains can be divided into two functional classes: (i) in smooth/non-muscle myosins, phosphorylation of the light chains by a calcium/calmodulin-dependent kinase regulates both interaction of the myosin head with actin and assembly of the myosin into filaments, (ii) the light chains of skeletal muscle myosins are similarly phosphorylated, but they play no apparent role in regulation. To discover the basis for the difference in regulatory properties of these two classes of light chains, we have synthesized in Escherichia coli, chimeric mutants composed of subdomains derived from the regulatory light chains of chicken skeletal and smooth muscle myosins. The regulatory capability of these mutants was analysed by their ability to regulate molluscan myosin. Using this test system, we identified the third subdomain of the regulatory light chain as being responsible for controlling not only the actin-myosin interaction, but also myosin filament assembly.

Amino Acid Sequence↗

PubMed1992

Role of the COOH-terminal nonhelical tailpiece in the assembly of a vertebrate nonmuscle myosin rod.

A short nonhelical sequence at the COOH-terminus of vertebrate nonmuscle myosin has been shown to enhance myosin filament assembly. We have analyzed the role of this sequence in chicken intestinal epithelial brush border myosin, using protein engineering/site-directed mutagenesis. Clones encoding the rod region of this myosin were isolated and sequenced. They were truncated at various restriction sites and expressed in Escherichia coli, yielding a series of mutant myosin rods with or without the COOH-terminal tailpiece and with serial deletions from their NH2-termini. Deletion of the 35 residue COOH-terminal nonhelical tailpiece was sufficient to increase the critical concentration for myosin rod assembly by 50-fold (at 150 mM NaCl, pH 7.5), whereas NH2-terminal deletions had only minor effects. The only exception was the longest NH2-terminal deletion, which reduced the rod to 119 amino acids and rendered it assembly incompetent. The COOH-terminal tailpiece could be reduced by 15 amino acids and it still efficiently promoted assembly. We also found that the tailpiece promoted assembly of both filaments and segments; assemblies which have different molecular overlaps. Rod fragments carrying the COOH-terminal tailpiece did not promote the assembly of COOH-terminally deleted material when the two were mixed together. The tailpiece sequence thus has profound effects on assembly, yet it is apparently unstructured and can be bisected without affecting its function. Taken together these observations suggest that the nonhelical tailpiece may act sterically to block an otherwise dominant but unproductive molecular interaction in the self assembly process and does not, as has been previously thought, bind to a specific target site(s) on a neighboring molecule.

Amino Acid Sequence↗

PubMed1991

Chimaeric myosin regulatory light chains: sub-domain switching experiments to analyse the function of the N-terminal EF hand.

The regulatory light chains (RLCs) located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation signals. The RLCs belong to a family of calcium binding proteins and are composed of four "EF hand" ancestral calcium binding motifs (numbered I to IV). To determine the role of the first EF hand (EF hand I) in the regulatory process, chimaeric light chains were constructed by protein engineering, by switching this region between smooth muscle and skeletal muscle myosin RLCs. For example, chimaera G(I)S consisted of EF hand I of the smooth muscle (gizzard) RLC and EF hands II to IV of the skeletal muscle RLC, whereas chimaera S(I)G consisted of EF hand I of the skeletal muscle RLC and EF hands II to IV of the smooth muscle RLC. The chimaeric RLCs were expressed in Escherichia coli using the pLcII expression system, and after isolation and purification their regulatory properties were compared with those of wild-type smooth and skeletal muscle myosin RLCs. The chimaeric RLCs bound to the myosin heads in scallop striated muscle myofibrils from which the endogenous RLCs had been removed ("desensitized" myofibrils) with similar affinities to those of the wild-type smooth and skeletal muscle RLCs. Both chimaeric RLCs were able to regulate the actin-activated Mg(2+)-ATPase activity of scallop myosin: G(I)S inhibited the ATPase in the presence and absence of Ca2+, like the wild-type skeletal muscle RLC, while S(I)G inhibited the myosin ATPase in the absence of Ca2+, and this inhibition was relieved on Ca2+ addition, in the same way as the wild-type smooth muscle RLC. Thus the type of regulation that the RLCs confer on the myosin is determined by the source of EF hands II to IV rather than that of EF hand I.

Amino Acid Sequence↗

PubMed1991

A folded (10 S) conformer of myosin from a striated muscle and its implications for regulation of ATPase activity.

Myosin from the striated adductor muscle of the scallop Pecten maximus is shown to fold into a compact 10 S conformer under relaxing conditions, as has been characterized for smooth and non-muscle myosins. The folding transition is accompanied by the trapping of nucleotide at the active site to give a species with a half-life of about an hour at 20 degrees C. Ca2+ binding to the specific, regulatory sites on a myosin head promotes unfolding to the extended 6 S conformer and activates product release by 60-fold. The unfolding transition, however, remains much slower than the contraction-relaxation cycle of scallop striated muscle and could not play a role in the regulation of these events. The dissociation of products from myosin heads in native thick filaments is Ca2(+)-regulated, but under relaxing conditions the nucleotide is released at least an order of magnitude faster than from the 10 S monomeric myosin, at a rate similar to that observed with heavy meromyosin. Thus, there is no evidence for any intermolecular interaction between neighbouring molecules in the filament analogous to the head-neck intramolecular interaction in the 10 S conformer. It is possible that the 10 S myosin state represents an inert form involved in the control of filament assembly during muscle growth and development. Removal of regulatory light chains or labelling the reactive heavy chain thiol of myosin prevents, or at least disfavours, formation of the folded 10 S conformer and allows separation of the modified protein from the native molecules.

Adenosine Triphosphate↗

PubMed1991

A nucleation--elongation mechanism for the self-assembly of side polar sheets of smooth muscle myosin.

Self-assembled filaments of smooth muscle myosin were observed by low dose electron microscopy to be flat side-polar sheets, in which the component molecules appeared straight and close-packed. Fraying experiments released small oligomers, in which molecules were staggered in parallel by about +/- 14 nm relative to two immediate neighbours, and were bound also to an antiparallel partner via a approximately 14 nm overlap at the very tip of the tail. We suggest a filament model which preserves these packing relationships. Adding stoichiometric amounts of MgATP to the filaments caused them to disassemble completely by progressive loss of material from their ends, at a limiting rate equivalent to about 2 monomers per second per end in physiological saline. The rate of the competing association reaction varied linearly with the monomer concentration, as determined in pressure-jump experiments. This suggests that myosin monomers, rather than dimers or higher oligomers, are the building blocks of these filaments. Shearing and annealing of assembled filaments appeared negligible on a time scale of a few hours. In consequence, filament number and filament length were dependent on the rate at which monomers were supplied to the assembly reaction, and on the number of filaments already present at the start of the assembly reaction.

Actin Cytoskeleton↗

PubMed1991

X-ray diffraction study of the structural changes accompanying phosphorylation of tarantula muscle.

Electron microscopy of negatively stained isolated thick filaments of tarantula muscle has revealed that phosphorylation of myosin regulatory light chains is accompanied by a loss of the helical order of myosin heads. From equatorial X-ray diffraction patterns of tarantula muscles in the phosphorylated state we have detected a mass movement in the myosin filaments that supports this finding.

Actin Cytoskeleton↗

PubMed1991

Localization of myosin in the cytoskeleton of brush border cells using monoclonal antibodies and confocal laser-beam scanning microscopy.

Monoclonal antibodies binding to the rod portion of brush border myosin were used to localize myosin in chicken intestinal brush border cells by indirect immunofluorescence. Isolated cells, or cells still attached in a sheet, were analyzed by conventional epifluorescence microscopy, which showed that most of the immunoreactive myosin is localized in the apical brush border (terminal web), and in a basal region. In addition, a weak, diffuse granular and rod-like labeling was detected throughout the cell body. Using the laser-scanning confocal microscope (White et al., 1987), a more precise localization of the myosin within the terminal web and the cell body was obtained. In the terminal web, most of the myosin was concentrated in a circumferential ring, below the plasma membrane, and the remaining myosin was found in the inter-rootlet area. These two populations of myosin were topologically strictly related, since they were found in the same optical sections. In the cell body, as well as in the basal region, the myosin was found to be associated with the outer limiting membrane of the cell, in a cortical location, whereas essentially no myosin was detected in the cytoplasm.

Animals↗

PubMed1991

Recombinant DNA approaches to study the role of the regulatory light chains (RLC) using scallop myosin as a test system.

The ability to exchange reversibly the regulatory light chains (RLCs) from scallop myosin has provided us with a test system to probe the mechanisms of regulation mediated by the RLCs from vertebrate skeletal, vertebrate smooth and molluscan myosins. The cloning and expression of these RLCs, together with domain-swapping and site-directed mutagenesis approaches, has allowed us to explore further the mechanisms involved and identify the functional importance of specific regions of the RLC molecule; for example, the presence of a high affinity metal binding site in the N-terminal domain and its interaction with the intact C-terminal domains are required for regulation.

Amino Acid Sequence↗

PubMed1991

Self-assembly pathway of nonsarcomeric myosin II.

Cells need to control the location and timing of actomyosin-dependent force generation, and appear to do so in the first instance by regulating myosin filament self-assembly (Yumura and Fukui, 1985). The mechanism of the self-assembly is little understood. In vitro it is a true self-assembly, which requires a short domain at the C terminus of the myosin molecule. The availability of this domain appears suppressed by the folding of the molecule into a compact, looped state. In vitro, the rate at which these looped molecules unfold turns out to be a key determinant of filament number and filament length.

Actin Cytoskeleton↗

PubMed1991

Small angle X-ray scattering studies on myosin.

Small angle X-ray scattering (SAXS) is a potentially powerful method for obtaining structural information from biological molecules in solution. The use of this technique in the laboratory has hitherto been limited by the long exposures necessary to obtain patterns on photographic film. Multi-wire area detectors, due to their high efficiency and absence of noise, enable patterns to be collected much more rapidly, typically in 1-2 h for a typical protein using laboratory sources. This opens up the possibility of using the technique on a semi-routine basis for a wide variety of problems. We outline the use of SAXS to characterise a large conformational change of myosin.

Animals↗

PubMed1990

The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli.

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca2(+)-calmodulin and binding to actin, actin-tropomyosin, Ca2(+)-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca2(+)-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.

Animals↗

PubMed1990

Structural predictions for the central domain of dystrophin.

The amino acid sequence of dystrophin indicates that the molecule has globular N- and C-terminal domains separated by a long central rod domain. The central rod contains multiple repeats, about 100 amino acids long and of variable length. These diverge sufficiently in sequence that, in previous studies, only 14 of the most similar repeats have been aligned and analysed in any detail. We show here that a heptad pattern of hydrophobic residues is preserved across all repeats. Using the heptad pattern together with a consensus sequence template, we identified and aligned 25 repeats in the dystrophin rod sequence. Each repeat consists of a constant-length core helix of 54 residues, coupled via a short linker to a weakly conserved variable-length helix, and then via a second linker to the next core. The variable-length helix appears truncated in repeats 10 and 13 and extended in repeats 4 and 20. The extension of repeat 20 is particularly interesting since it corresponds to a hotspot of dystrophy-inducing mutations. Detailed modelling suggests that the classical Speicher-Marchesi [(1984) Nature 311, 177-180] model for spectrin may not be appropriate to dystrophin without some modification. We propose that whilst the repeating structural motif in dystrophin is probably a bead of triple coiled coil, this bead is twice as massive as, and out of phase with, those proposed for spectrin. Our model raises the possibility that the rod domain of dystrophin may confer elasticity on the molecule. Deletions which truncate this region would then reduce the extensibility of the molecule without affecting actin crosslinking, consistent with their typically producing the relatively benign Becker phenotype of muscular dystrophy.

Actinin↗

PubMed1989

Regulatory and essential light-chain-binding sites in myosin heavy chain subfragment-1 mapped by site-directed mutagenesis.

Site-directed mutagenesis of the cloned subfragment-1 (S-1) region of the unc-54 gene, encoding the myosin heavy chain B (MHC B) from Caenorhabditis elegans, has been used to locate binding sites for the regulatory and essential light chains. MHC B S-1 synthesized in Escherichia coli co-migrated with rabbit skeletal muscle myosin S-1 (Mr 90,000), was recognized by anti-nematode myosin antiserum on immunoblots, and specifically bound to 125I-labelled regulatory and essential light chains in a gel overlay assay. Deletion of 102 residues from the C terminus (mutant 655) reduced regulatory and essential light-chain binding to about 30% and 20% of wild-type levels, respectively. Similar reductions in relative binding of the two light chains were seen with mutant 534, in which 38 residues were deleted from the C terminus. Potential binding sites within 75 residues of the C terminus of S-1 were mapped by construction of five other mutant S-1 clones (398, 399, 400, 409 and 411) containing internal deletions of ten to 12 amino acid residues. These showed up to 30% reductions in their ability to bind essential light chains, but did not differ significantly from wild-type in their ability to bind regulatory light chains. Another mutant, 415, containing a deletion of a conserved acidic hexapeptide, E-D-I-R-D-E, showed enhancement of binding of regulatory and essential light chains to 150% and 165% of wild-type levels. Hence, the major binding sites for both light chains are within 38 amino acid residues of the C terminus.

Amino Acid Sequence↗

PubMed1989

Parallel modulation of brush border myosin conformation and enzyme activity induced by monoclonal antibodies.

Monoclonal antibodies binding to distinct epitopes on the tail of brush border myosin were used to modulate the conformation and state of assembly of this myosin. BM1 binds 1:3 of the distance from the tip of the tail to the head and prevents the extended-tail (6S) monomer from folding into the assembly-incompetent folded-tail (10S) state, whereas BM4 binds to the tip of the myosin tail, and induces the myosin to fold into the 10S state. Thus, at physiological ionic strength BM1 promotes and BM4 blocks the assembly of the myosin into filaments. Using BM1 and BM4 together, we were able to prevent both folding and filament assembly, thus locking myosin molecules in the extended-tail 6S monomer conformation at low ionic strength where they normally assemble into filaments. Using these myosin-antibody complexes, we were able to investigate independently the effects of folding of the myosin tail and assembly into filaments on the myosin MgATPase. The enzymatic activities were measured from the fluorescent profiles during the turnover of the ATP analogue formycin triphosphate (FTP). Extended-tail (6S) myosin molecules had an FTPase activity of 1-5 X 10(-3) s-1, either at high ionic strength as a monomer alone or when complexed with antibody, or at low ionic strength as filaments or when maintained as extended-tail monomers by the binding of BM1 and BM4. Folding of the molecules into the 10S state reduced this rate by an order of magnitude, effectively trapping the products of FTP hydrolysis in the active sites.

Animals↗