Kevin Leonard

Evidence that nebulin is a protein-ruler in muscle thin filaments

Partial amino acid sequence was obtained from the massive myofibrillar protein nebulin. This consists of repeating motifs of about 35 residues and super‐repeats of 7 × 35 = 245 residues. The repeat‐motifs are likely to be largely α‐helical and to interact with both actin and tropomyosin in thin filaments. Nebulin from different species was found to vary in size in proportion to filament length. The data are consistent with the proposal that nebulin acts as a protein‐ruler to regulate precise thin filament assembly.

Solution structure of polyglutamine tracts in GST-polyglutamine fusion proteins.

Aggregation of expanded polyglutamine (polyQ) seems to be the cause of various genetic neurodegenerative diseases. Relatively little is known as yet about the polyQ structure and the mechanism that induces aggregation. We have characterised the solution structure of polyQ in a proteic context using a model system based on glutathione S‐transferase fusion proteins. A wide range of biophysical techniques was applied. For the first time, nuclear magnetic resonance was used to observe directly and selectively the conformation of polyQ in the pathological range. We demonstrate that, in solution, polyQs are in a random coil conformation. However, under destabilising conditions, their aggregation behaviour is determined by the polyQ length.

Three-dimensional structure of ubiquinol: Cytochrome C reductase from Neurospora mitochondria determined by electron microscopy of membrane crystals

Abstract Membrane crystals have been prepared from mitochondrial ubiquinol: cytochrome c reductase by mixing the enzyme-Triton complex with phospholipid-Triton micelles and subsequently removing the Triton. The electron micrographs of the negatively stained crystals diffract to 2·5 nm, with unit cell dimensions of 13·7 nm by 17·4 nm. The enzyme is arranged in a two-sided plane group P 22 1 2 1 , i.e. alternate molecules span the bilayer in an up and down manner. By combining tilted views of the membrane crystals, a low-resolution three-dimensional structure of the enzyme has been calculated. The structure shows that the enzyme is a dimer, the monomers being related by a 2-fold axis running perpendicular to the membrane. The monomeric units of the enzyme are elongated, extending approximately 15 nm across the membrane. The protein is unequally distributed with about 30% of the total mass located in the bilayer, 50% in a section which extends 7 nm from one side of the bilayer and 20% in a section which extends 3 nm from the opposite side of the bilayer. The two monomeric units are in contact only in the membraneous section. This structure is compared with a model of the enzyme which is derived from biochemical properties of the isolated subunits.

Troponin of asynchronous flight muscle

Troponin has been prepared from the asynchronous flight muscle of Lethocerus (water bug) taking special care to prevent proteolysis. The regulatory complex contained tropomyosin and troponin components. The troponin components were Tn-C (18,000 Mr), Tn-T (apparent Mr 53,000) and a heavy component, Tn-H (apparent Mr 80,000). The troponin was tightly bound to tropomyosin and could not be dissociated from it in non-denaturing conditions. A complex of Tn-T, Tn-H and tropomyosin inhibited actomyosin ATPase activity and the inhibition was relieved by Tn-C from vertebrate striated muscle in the presence of Ca2+. However, unlike vertebrate Tn-I, Tn-H by itself was not inhibitory. Monoclonal antibodies were obtained to Tn-T and Tn-H. Antibody to Tn-T was used to screen an expression library of Drosophila cDNA cloned in lambda phage. The sequence of cDNA coding for the protein was determined and hence the amino acid sequence. The Drosophila protein has a sequence similar to that of vertebrate skeletal and cardiac Tn-T. The sequence extends beyond the carboxyl end of the vertebrate sequences, and the last 40 residues are acidic. Part of the sequence of Drosophila Tn-T is homologous to the carboxyl end of the Drosophila myosin light chain MLC-2 and one anti-Tn-T antibody cross-reacted with the light chain. Lethocerus Tn-H is related to the large tropomyosins of Drosophila flight muscle, for which the amino acid sequence is known, since antibodies that recognize this component also recognize the large tropomyosins. Tn-H is easily digested by calpain, suggesting that part of the molecule has an extended configuration. Electron micrographs of negatively stained specimens showed that Lethocerus thin filaments have projections at about 39 nm intervals, which are not seen on thin filaments from vertebrate striated muscle and are probably due to the relatively large troponin complex. Decoration of the thin filaments with myosin subfragment-1 in rigor conditions appeared not to be affected by the troponin. The troponin of asynchronous flight muscle lacks the Tn-I component of vertebrate striated muscle. Tn-H occurs only in the flight muscle and may be involved in the activation of this muscle by stretch.

Phosphorylation of KSP motifs in the C-terminal region of titin in differentiating myoblasts.

Titin is a giant structural protein of striated muscle (M(r) approximately 3000 kDa) and single molecules span sarcomeres from the M‐ to Z‐lines. We have cloned and sequenced the C‐terminal region of the titin molecule, which is an integral part of M‐lines and forms intimate contacts with the 165 and 190 kDa M‐line proteins. In contrast to the regular motif patterns of the A‐band portion of titin, the 5.7 kb of titin sequences from the M‐line show a complex structure of immunoglobulin‐C2 repeats, separated by unique interdomain insertion sequences. As a striking feature, one interdomain insertion comprises four KSP repeats analogous to the multi‐phosphorylation repeats of neurofilament subunits H and M. In vitro phosphorylation assays with expressed titin KSP sequences detect high levels of titin KSP phosphorylating kinases in developing but not in differentiated muscle. Since this kinase activity can be depleted from myocyte extracts by antibodies against cdc2 kinase and p13suc1 beads, the titin KSP kinase is structurally related to cdc2 kinase. We suggest that titin C‐terminal phosphorylation by SP‐specific kinases is regulated during differentiation, and that this may control the assembly of M‐line proteins into regular structures during myogenesis.

The aerolysin membrane channel is formed by heptamerization of the monomer.

The cytolytic toxin aerolysin has been found to form heptameric oligomers by SDS‐PAGE electrophoresis, STEM mass measurements of single oligomers and image analysis of two‐dimensional membrane crystals. Two types of crystal, flat sheets and long regular tubes, have been obtained by reconstitution of purified protein and Escherichia coli phospholipids. A noise‐filtered image of the best crystalline sheets reveals a structure with 7‐fold symmetry containing a central strongly stain‐excluding ring that encircles a dark stain‐filled channel 17 A in diameter. The ring is surrounded by seven arms each made up of two unequal sized domains. By combining projected views and side‐views, a simplified model of the aerolysin channel complex has been constructed. The relevance of this structure to the mode of action of aerolysin is discussed.

Identification and localization of high molecular weight proteins in insect flight and leg muscle.

Thick and thin filaments in asynchronous flight muscle overlap nearly completely and thick filaments are attached to the Z‐disc by connecting filaments. We have raised antibodies against a fraction of Lethocerus flight muscle myofibrils containing Z‐discs and associated filaments and also against a low ionic strength extract of myofibrils. Monoclonal antibodies were obtained to proteins of 800 kd (p800), 700 kd (p700), 400 kd (p400) and alpha‐actinin. The positions of the proteins in Lethocerus flight and leg myofibrils were determined by immunofluorescence and electron microscopy. p800 is in connecting filaments of flight myofibrils and in A‐bands of leg myofibrils. p700 is in Z‐discs of flight myofibrils and an immunologically related protein, p500, is in leg muscle Z‐discs. p400 is in M‐lines of both flight and leg myofibrils. Preliminary DNA sequencing shows that p800 is related to vertebrate titin and nematode twitchin. Molecules of p800 could extend from the Z‐disc a short way along thick filaments, forming a mechanical link between the two structures. All three high molecular weight proteins probably stabilize the structure of the myofibril.

Kettin, a large modular protein in the Z-disc of insect muscles.

Z‐discs of insect flight muscle contain a large protein of 500–700 kDa. Monoclonal antibodies label an epitope in the molecule at the Z‐disc in Drosophila and Lethocerus (waterbug). A partial cDNA of 1.6 kb from the Drosophila gene has been cloned and sequenced. The corresponding amino acid sequence has a modular structure composed of four conserved repeats of 95 amino acids homologous to immunoglobulin C2 domains (called class II domains in muscle proteins), separated by less conserved linker sequences of 35 amino acids. An expressed class II domain with flanking linker sequences binds to actin and alpha‐actinin but not to myosin. Single molecules of the protein would be large enough to span the Z‐disc. We suggest that the protein acts as scaffolding in the Z‐disc and we call the protein kettin. The Ca2+ activated protease, calpain, disrupts the Z‐disc of striated muscle, releasing alpha‐actinin intact. Calpain digests kettin to a series of peptides of between 30 and 170 kDa which are released from the myofibril. Digestion of kettin may cause disintegration of the Z‐disc and alpha‐actinin release which lead to disassembly of the myofibril.

Structure of a Drosophila sigma class glutathione S-transferase reveals a novel active site topography suited for lipid peroxidation products

Insect glutathione-S-transferases (GSTs) are grouped in three classes, I, II and recently III; class I (Delta class) enzymes together with class III members are implicated in conferring resistance to insecticides. Class II (Sigma class) GSTs, however, are poorly characterized and their exact biological function remains elusive. Drosophila glutathione S-transferase-2 (GST-2) (DmGSTS1-1) is a class II enzyme previously found associated specifically with the insect indirect flight muscle. It was recently shown that GST-2 exhibits considerable conjugation activity for 4-hydroxynonenal (4-HNE), a lipid peroxidation product, raising the possibility that it has a major anti-oxidant role in the flight muscle. Here, we report the crystal structure of GST-2 at 1.75A resolution. The GST-2 dimer shows the canonical GST fold with glutathione (GSH) ordered in only one of the two binding sites. While the GSH-binding mode is similar to other GST structures, a distinct orientation of helix alpha6 creates a novel electrophilic substrate-binding site (H-site) topography, largely flat and without a prominent hydrophobic-binding pocket, which characterizes the H-sites of other GSTs. The H-site displays directionality in the distribution of charged/polar and hydrophobic residues creating a binding surface that explains the selectivity for amphipolar peroxidation products, with the polar-binding region formed by residues Y208, Y153 and R145 and the hydrophobic-binding region by residues V57, A59, Y211 and the C-terminal V249. A structure-based model of 4-HNE binding is presented. The model suggest that residues Y208, R145 and possibly Y153 may be key residues involved in catalysis.

Crystallization of p68 on lipid monolayers and as three-dimensional single crystals

Two-dimensional crystals of p68, a Ca2+ -binding protein that has homology with members of the lipocortin/calpactin family, were obtained by interaction with a phospholipid monolayer. By measuring surface pressure at constant surface area, p68 was found to interact in a Ca2+ -dependent manner specifically with phosphatidylethanolamine, less so with phosphatidylserine and not at all with phosphatidylcholine. With dimyristoyl-phosphatidylethanolamine, two-dimensional crystalline arrays were formed. Image analysis of electron micrographs of these crystals, which diffracted to about 50 A, revealed p3 symmetry with a unit cell of about 178 A by 178 A; the protein densities showed a two-domain structure giving a cylindrical molecule of about 100 A by 35 A diameter packed as trimers. Three-dimensional microcrystals obtained without lipid or Ca2+ were suitable for electron microscopy and gave a tetragonal unit cell of about 256 A by 68 A. The implications of these observations on the structure and lipid specificity of p68 binding are discussed.