Molecular mechanism of interactions between ACAD9 and binding partners in mitochondrial respiratory complex I assembly.

The dual function protein ACAD9 catalyzes α,β-dehydrogenation of fatty acyl-CoA thioesters in fatty acid β-oxidation and is an essential chaperone for mitochondrial respiratory complex I (CI) assembly. ACAD9, ECSIT, and NDUFAF1 interact to form the core mitochondrial CI assembly complex. Current studies examine the molecular mechanism of ACAD9/ECSIT/NDUFAF1interactions.

Crystal structure of human mitochondrial trifunctional protein, a fatty acid β-oxidation metabolon.

Membrane-bound mitochondrial trifunctional protein (TFP) catalyzes β-oxidation of long chain fatty acyl-CoAs, employing 2-enoyl-CoA hydratase (ECH), 3-hydroxyl-CoA dehydrogenase (HAD), and 3-ketothiolase (KT) activities consecutively. Inherited deficiency of TFP is a recessive genetic disease, manifesting in hypoketotic hypoglycemia, cardiomyopathy, and sudden death. We have determined the crystal structure of human TFP at 3.

Enhancing the protective immune response against botulism.

The need for a vaccine against botulism has increased since the discontinuation of the pentavalent (ABCDE) botulinum toxoid vaccine by the Centers for Disease Control and Prevention. The botulinum toxins (BoNTs) are the primary virulence factors and vaccine components against botulism. BoNTs comprise three domains which are involved in catalysis (LC), translocation (HCT), and host receptor binding (HCR).

Botulinum neurotoxin serotype C associates with dual ganglioside receptors to facilitate cell entry.

Background: How botulinum neurotoxin serotype C (BoNT/C) enters neurons is unclear.

Results: BoNT/C utilizes dual gangliosides as host cell receptors.

Conclusion: BoNT/C accesses gangliosides on the plasma membrane.

Unique ganglioside recognition strategies for clostridial neurotoxins.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin are the causative agents of the paralytic diseases botulism and tetanus, respectively. The potency of the clostridial neurotoxins (CNTs) relies primarily on their highly specific binding to nerve terminals and cleavage of SNARE proteins. Although individual CNTs utilize distinct proteins for entry, they share common ganglioside co-receptors.

Novel ganglioside-mediated entry of botulinum neurotoxin serotype D into neurons.

Botulinum Neurotoxins (BoNTs) are organized into seven serotypes, A-G. Although several BoNT serotypes enter neurons through synaptic vesicle cycling utilizing dual receptors (a ganglioside and a synaptic vesicle-associated protein), the entry pathway of BoNT/D is less well understood. Although BoNT/D entry is ganglioside-dependent, alignment and structural studies show that BoNT/D lacks key residues within a conserved ganglioside binding pocket that are present in BoNT serotypes A, B, E, F, and G, which indicate that BoNT/D-ganglioside interactions may be unique.

Unique ganglioside binding by botulinum neurotoxins C and D-SA.

The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G), based on a lack of cross-antiserum neutralization. The BoNT/C and BoNT/D serotypes include mosaic toxins that are organized as D-C and C-D toxins.

Identification of a unique ganglioside binding loop within botulinum neurotoxins C and D-SA .

The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G) based on a lack of cross antiserum neutralization. BoNTs utilize gangliosides as components of the host receptors for binding and entry into neurons.

Functional insights into human HMG-CoA lyase from structures of Acyl-CoA-containing ternary complexes.

HMG-CoA lyase (HMGCL) is crucial to ketogenesis, and inherited human mutations are potentially lethal. Detailed understanding of the HMGCL reaction mechanism and the molecular basis for correlating human mutations with enzyme deficiency have been limited by the lack of structural information for enzyme liganded to an acyl-CoA substrate or inhibitor. Crystal structures of ternary complexes of WT HMGCL with the competitive inhibitor 3-hydroxyglutaryl-CoA and of the catalytically deficient HMGCL R41M mutant with substrate HMG-CoA have been determined to 2.

Gangliosides as high affinity receptors for tetanus neurotoxin.

Tetanus neurotoxin (TeNT) is an exotoxin produced by Clostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a approximately 150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T).

Glycosylated SV2 and gangliosides as dual receptors for botulinum neurotoxin serotype F.

Botulinum neurotoxin causes rapid flaccid paralysis through the inhibition of acetylcholine release at the neuromuscular junction. The seven BoNT serotypes (A-G) have been proposed to bind motor neurons via ganglioside-protein dual receptors. To date, the structure-function properties of BoNT/F host receptor interactions have not been resolved.

Human mevalonate diphosphate decarboxylase: characterization, investigation of the mevalonate diphosphate binding site, and crystal structure.

Expression in Escherichia coli of his-tagged human mevalonate diphosphate decarboxylase (hMDD) has expedited enzyme isolation, characterization, functional investigation of the mevalonate diphosphate binding site, and crystal structure determination (2.4A resolution). hMDD exhibits V(max)=6.

Biochemical and structural basis for feedback inhibition of mevalonate kinase and isoprenoid metabolism.

Mevalonate kinase (MK), which catalyzes a key reaction in polyisoprenoid and sterol metabolism in many organisms, is subject to feedback regulation by farnesyl diphosphate and related compounds. The structures of human mevalonate kinase and a binary complex of the rat enzyme incubated with farnesyl thiodiphosphate (FSPP) are reported. Significant FSPP hydrolysis occurs under crystallization conditions; this results in detection of farnesyl thiophosphate (FSP) in the structure of the binary complex.

The effect of a Glu370Asp mutation in glutaryl-CoA dehydrogenase on proton transfer to the dienolate intermediate.

We have determined steady-state rate constants and net rate constants for the chemical steps in the catalytic pathway catalyzed by the E370D mutant of glutaryl-CoA dehydrogenase and compared them with those of the wild-type dehydrogenase. We sought rationales for changes in these rate constants in the structure of the mutant cocrystallized with the alternate substrate, 4-nitrobutyric acid. Substitution of aspartate for E370, the catalytic base, results in a 24% decrease in the rate constant for proton abstraction at C-2 of 3-thiaglutaryl-CoA as the distance between C-2 of the ligand and the closest carboxyl oxygen at residue 370 increases from 2.

Light chain of botulinum neurotoxin serotype A: structural resolution of a catalytic intermediate.

Botulinum neurotoxin serotype A (BoNT/A, 1296 residues) is a zinc metalloprotease that cleaves SNAP25 to inhibit the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. BoNT/A is a disulfide-linked di-chain protein composed of an N-terminal, thermolysin-like metalloprotease light chain domain (LC/A, 448 residues) and a C-terminal heavy chain domain (848 residues) that can be divided into two subdomains, a translocation subdomain and a receptor binding subdomain. LC/A cleaves SNAP25 between residues Gln197-Arg198 and, unlike thermolysin, recognizes an extended region of SNAP25 for cleavage.

Crystal structure of human 3-hydroxy-3-methylglutaryl-CoA Lyase: insights into catalysis and the molecular basis for hydroxymethylglutaric aciduria.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the ketogenic pathway that supplies metabolic fuel to extrahepatic tissues. Enzyme deficiency may be due to a variety of human mutations and can be fatal. Diminished activity has been explained based on analyses of recombinant human mutant proteins or, more recently, in the context of structural models for the enzyme.

Crystal structures of two bacterial 3-hydroxy-3-methylglutaryl-CoA lyases suggest a common catalytic mechanism among a family of TIM barrel metalloenzymes cleaving carbon-carbon bonds.

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.

Crystal structure of the interferon-induced ubiquitin-like protein ISG15.

The biological effects of the ISG15 protein arise in part from its conjugation to cellular targets as a primary response to interferon-alpha/beta induction and other markers of viral or parasitic infection. Recombinant full-length ISG15 has been produced for the first time in high yield by mutating Cys78 to stabilize the protein and by cloning in a C-terminal arginine cap to protect the C terminus against proteolytic inactivation. The cap is subsequently removed with carboxypeptidase B to yield mature biologically active ISG15 capable of stoichiometric ATP-dependent thiolester formation with its human UbE1L activating enzyme.

Crystal structures of human glutaryl-CoA dehydrogenase with and without an alternate substrate: structural bases of dehydrogenation and decarboxylation reactions.

Acyl-CoA dehydrogenases (ACDs) are a family of flavoenzymes that metabolize fatty acids and some amino acids. Of nine known ACDs, glutaryl-CoA dehydrogenase (GCD) is unique: in addition to the alpha,beta-dehydrogenation reaction, common to all ACDs, GCD catalyzes decarboxylation of glutaryl-CoA to produce CO(2) and crotonyl-CoA. Crystal structures of GCD and its complex with 4-nitrobutyryl-CoA have been determined to 2.

The structure of a binary complex between a mammalian mevalonate kinase and ATP: insights into the reaction mechanism and human inherited disease.

Mevalonate kinase catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate, a key intermediate in the pathways of isoprenoids and sterols. Deficiency in mevalonate kinase activity has been linked to mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS). The crystal structure of rat mevalonate kinase in complex with MgATP has been determined at 2.