Ivan Rayment

Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle

The muscle myosins are hexomeric proteins consisting of two heavy chains and two pairs of light chains, the latter called essential (ELC) and regulatory (RLC). The light chains stabilize the long alpha helical neck of the myosin head. Their function in striated muscle, however, is only partially understood. We report here the identification of distinct missense mutations in a skeletal/ventricular ELC and RLC, each of which are associated with a rare variant of cardiac hypertrophy as well as abnormal skeletal muscle.We show that myosin containing the mutant ELC has abnormal function, map the mutant residues on the three–dimensional structure of myosin and suggest that the mutations disrupt the stretch activation response of the cardiac papillary muscles.

X-ray structure of the magnesium(II).ADP.vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 A resolution.

The structure of the vanadate-trapped ADP complex of a truncated head of Dictyostelium myosin II consisting of residues Asp 2-Asn 762 has been determined by molecular replacement at 1.9 A resolution and refined to a crystallographic R-factor of 19.4%. The crystals belong to the orthorhombic space group C2221 where a = 84.50 A, b = 145.4 A, and c = 152.8 A. The conformation of the protein is similar to that of MgADP.AlF4.SlDc [Fisher, A.J., et al. (1995) Biochemistry 34, 8960-8972]. The nucleotide binding site contains a complex between MgADP and vanadate where MgADP exhibits a very similar conformation to that seen in previous complexes. The vanadate ion adopts a trigonal bipyramidal coordination. The three equatorial oxygen ligands are fairly short, average 1.7 A, relative to a single bond distance of approximately 1.8 A and are coordinated to the magnesium ion, N zeta of Lys 185, and five other protein ligands. The apical coordination to the vanadate ion is filled by a terminal oxygen on the beta-phosphate of ADP and a water molecule at bond distances of 2.1 and 2.3 A, respectively. The long length of the apical bonds suggests that the bond order is considerably less than unity. This structure confirms the earlier suggestion that vanadate is a model for the transition state of ATP hydrolysis and thus provides insight into those factors that are responsible for catalysis. In particular, it shows that the protein ligands and water structure surrounding the gamma-phosphate pocket are oriented to stabilize a water molecule in an appropriate position for in-line nucleophilic attack on the gamma-phosphorus of ATP. This structure reveals also an orientation of the COOH-terminal region beyond Thr 688 which is very different from that observed in either MgADP.BeFx.SlDc or chicken skeletal myosin subfragment 1. This is consistent with the COOH-terminal region of the molecule playing an important role in the transduction of chemical energy of hydrolysis of ATP into mechanical movement.

Structure and Function of Enzymes of the Leloir Pathway for Galactose Metabolism

In most organisms, the conversion of -D-galactose to the more metabolically useful glucose 1-phosphate is accomplished by the action of four enzymes that constitute the Leloir pathway (Scheme 1). In the first step of this pathway, -D-galactose is epimerized to -D-galactose by galactose mutarotase. The next step involves the ATP-dependent phosphorylation of -D-galactose by galactokinase to yield galactose 1-phosphate. As indicated in Scheme 1, the third enzyme in the pathway, galactose-1-phosphate uridylyltransferase, catalyzes the transfer of a UMP group from UDP-glucose to galactose 1-phosphate, thereby generating glucose 1-phosphate and UDP-galactose. To complete the pathway, UDP-galactose is converted to UDP-glucose by UDP-galactose 4-epimerase. In humans, defects in the genes encoding for galactokinase, uridylyltransferase, or epimerase can give rise to the diseased state referred to collectively as galactosemia (1, 2). Although galactosemia is rare, it is potentially lethal with clinical manifestations including intellectual retardation, liver dysfunction, and cataract formation, among others. Indeed, the enzymes of the Leloir pathway have attracted significant research attention for well over 30–40 years, in part because of their important metabolic role in normal galactose metabolism. As of this year, the three-dimensional structures of all of the enzymes of the Leloir pathway have now been defined. It is thus timely to present in this minireview recent advances in our understanding of the structure and function of these enzymes. For a discussion of the literature prior to 1996, see Ref. 3.

The structural basis of blebbistatin inhibition and specificity for myosin II.

Molecular motors play a central role in cytoskeletal-mediated cellular processes and thus present an excellent target for cellular control by pharmacological agents. Yet very few such compounds have been found. We report here the structure of blebbistatin, which inhibits specific myosin isoforms, bound to the motor domain of Dictyostelium discoideum myosin II. This reveals the structural basis for its specificity and provides insight into the development of new agents.

Structure, mechanism and regulation of pyruvate carboxylase

PC (pyruvate carboxylase) is a biotin-containing enzyme that catalyses the HCO(3)(-)- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the tricarboxylic acid cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis or lipogenesis and in insulin secretion. The enzyme was discovered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC from most organisms is a tetrameric protein that is allosterically regulated by acetyl-CoA and aspartate. High-resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details of the binding sites and the relative positions of the biotin carboxylase, carboxyltransferase and biotin carboxyl carrier domains, and also a unique allosteric effector domain. In the presence of the allosteric effector, acetyl-CoA, the biotin moiety transfers the carboxy group between the biotin carboxylase domain active site on one polypeptide chain and the carboxyltransferase active site on the adjacent antiparallel polypeptide chain. In addition, the bona fide role of PC in the non-gluconeogenic tissues has been studied using a combination of classical biochemistry and genetic approaches. The first cloning of the promoter of the PC gene in mammals and subsequent transcriptional studies reveal some key cognate transcription factors regulating tissue-specific expression. The present review summarizes these advances and also offers some prospects in terms of future directions for the study of this important enzyme.

Active site comparisons highlight structural similarities between myosin and other P-loop proteins

The phosphate binding loop (P-loop) is a common feature of a large number of enzymes that bind nucleotide whose consensus sequence is often used as a fingerprint for identifying new members of this group. We review here the binding sites of nine purine nucleotide binding proteins, with a focus on their relationship to the active site of myosin. This demonstrates that there is considerable conversation in the distribution and nature of the ligands that coordinate the triphosphate moiety. This comparison further suggests that at least myosin and the G-proteins utilize a similar mechanism for nucleotide hydrolysis.

Conservation within the myosin motor domain: implications for structure and function.

BACKGROUND Myosins are motors that use energy supplied by ATP to travel along actin filaments. The structure of myosin is known, but the actin-binding site is not well defined, and the mechanisms by which actin activates ATP hydrolysis by myosin, and myosin moves relative to the actin filament, developing force, are not fully understood. Previous phylogenetic analyses of the motor domain of myosins have identified up to twelve classes. We set out to analyse the positions of conserved residues within this domain in detail, and relate the conserved residues to the myosin structure. RESULTS Our analysis indicates that there are at least thirteen myosin classes. Conserved residues in the motor domain have been positioned within the framework provided by the recent crystal structures, thus helping to define those residues involved in actin and ATP binding, in hydrolysis and in conformational change. This has revealed remarkably poor overall conservation at the site thought to be involved in actin binding, but several highly conserved residues have been identified that may be functionally important. CONCLUSIONS Information from such a sequence analysis is a useful tool in the further interpretation of X-ray structures. It allows the position of crucial residues from other members of a superfamily to be determined within the framework provided by the known structures and the functional significance of conserved or mutated residues to be assessed.

The molecular structure of insecticyanin from the tobacco hornworm Manduca sexta L. at 2.6 A resolution.

Insecticyanin, a blue biliprotein isolated from the tobacco hornworm Manduca sexta L., is involved in insect camouflage. Its three‐dimensional structure has now been solved to 2.6 A resolution using the techniques of multiple isomorphous replacement, non‐crystallographic symmetry averaging about a local 2‐fold rotation axis and solvent flattening. All 189 amino acids have been fitted to the electron density map. The map clearly shows that insecticyanin is a tetramer with one of its molecular 2‐fold axes coincident to a crystallographic dyad. The individual subunits have overall dimensions of 44 A X 37 A X 40 A and consist primarily of an eight‐stranded anti‐parallel beta‐barrel flanked on one side by a 4.5‐turn alpha‐helix. Interestingly the overall three‐dimensional fold of the insecticyanin subunit shows remarkable similarity to the structural motifs of bovine beta‐lactoglobulin and the human serum retinol‐binding protein. The electron density attributable to the chromophore is unambiguous and shows that it is indeed the gamma‐isomer of biliverdin. The biliverdin lies towards the open end of the beta‐barrel with its two propionate side chains pointing towards the solvent and it adopts a rather folded conformation, much like a heme.

[12] Reductive alkylation of lysine residues to alter crystallization properties of proteins

Publisher Summary This chapter discusses the reductive alkylation of lysine residues to alter the crystallization properties of proteins. Chemical modification has played an essential role in the development of protein function. The reductive alkylation of lysine residues involves the initial formation of a Schiff base between the ɛ-amino group of a lysine residue and a ketone or aldehyde that is then reduced to a secondary or tertiary amine. In principle, a wide variety of alkyl moieties can be added to an amino group by reductive alkylation. In practice, the majority of cases employing this chemical modification have focused on adding methyl groups using formaldehyde because of the greater reactivity of formaldehyde than other ketones or aldehydes and because this modification has the mildest effect on the biochemical properties of a protein. The protocols described in the chapter are designed for complete modification of all available lysine residues. Amino acid analysis is the best way of determining the degree of modification of the lysine residues after reductive alkylation. It has the advantage of quantitatively defining the number of non-, mono-, di-, and tri-methylated lysine residues. It reveals the presence of any side reactions with other reactive amino acid side chains in a protein.

Actin-targeting natural products: structures, properties and mechanisms of action

Abstract.Natural small-molecule inhibitors of actin cytoskeleton dynamics have long been recognized as valuable molecular probes for dissecting complex mechanisms of cellular function. More recently, their potential use as chemotherapeutic drugs has become a focus of scientific investigation. The primary focus of this review is the molecular mechanism by which different actin-targeting natural products function, with an emphasis on structural considerations of toxins for which high-resolution structural information of their interaction with actin is available. By comparing the molecular interactions made by different toxin families with actin, the structural themes of those that alter filament dynamics in similar ways can be understood. This provides a framework for novel synthetic-compound designs with tailored functional properties that could be applied in both research and clinical settings.