Biomedical subjects

David T Chuang

Publications and source records attributed to David T Chuang.

At least 19 recordsLinked to original sources

PubMed2025

BCKDHA-BCKDHB digenic gene therapy restores metabolic homeostasis in two mouse models and a calf with classic maple syrup urine disease.

Classic maple syrup urine disease (MSUD) results from biallelic mutations in genes that encode the branched-chain α-ketoacid dehydrogenase E1α (BCKDHA), E1β (BCKDHB), or dihydrolipoamide branched-chain transacylase (DBT) subunits, which interact to form the mitochondrial BCKDH complex that decarboxylates ketoacid derivatives of leucine, isoleucine, and valine. MSUD is an inborn error of metabolism characterized by recurrent life-threatening neurologic crises and progressive brain injury that can only be managed with an exacting prescription diet or allogeneic liver transplant. To develop a gene replacement therapy for MSUD, we designed a dual-function recombinant adeno-associated virus serotype 9 (rAAV9) vector to deliver codon-optimized BCKDHA and BCKDHB (rAAV9.hA-BiP-hB) to the liver, muscle, heart, and brain. rAAV9.hA-BiP-hB restored coexpression of BCKDHA and BCKDHB as well as BCKDH holoenzyme activity in BCKDHA-/- HEK293T cells and did not perturb physiologic branched-chain amino acid homeostasis in wild-type mice at a systemic dose of 2.7 × 1014 vector genomes per kilogram. In two models of severe MSUD (Bckdha-/- and Bckdhb-/- mice) and a newborn calf homozygous for BCKDHA c.248C>T, one postnatal injection prevented perinatal death, normalized growth, restored coordinated expression of BCKDHA and BCKDHB in the skeletal muscle, liver, heart, and brain, and stabilized MSUD biomarkers in the face of high protein ingestion. In summary, we developed a one-time BCKDHA-BCKDHB systemic dual-gene replacement strategy that holds promise as a therapeutic alternative to prescription diet and liver transplant for treatment of MSUD types 1A and 1B, the two most common forms of MSUD in humans.

Animals↗

PubMed2006

A synchronized substrate-gating mechanism revealed by cubic-core structure of the bovine branched-chain alpha-ketoacid dehydrogenase complex.

The dihydrolipoamide acyltransferase (E2b) component of the branched-chain alpha-ketoacid dehydrogenase complex forms a cubic scaffold that catalyzes acyltransfer from S-acyldihydrolipoamide to CoA to produce acyl-CoA. We have determined the first crystal structures of a mammalian (bovine) E2b core domain with and without a bound CoA or acyl-CoA. These structures reveal both hydrophobic and the previously unreported ionic interactions between two-fold-related trimers that build up the cubic core. The entrance of the dihydrolipoamide-binding site in a 30-A long active-site channel is closed in the apo and acyl-CoA-bound structures. CoA binding to one entrance of the channel promotes a conformational change in the channel, resulting in the opening of the opposite dihydrolipoamide gate. Binding experiments show that the affinity of the E2b core for dihydrolipoamide is markedly increased in the presence of CoA. The result buttresses the model that CoA binding is responsible for the opening of the dihydrolipoamide gate. We suggest that this gating mechanism synchronizes the binding of the two substrates to the active-site channel, which serves as a feed-forward switch to coordinate the E2b-catalyzed acyltransfer reaction.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2006

Structure of the subunit binding domain and dynamics of the di-domain region from the core of human branched chain alpha-ketoacid dehydrogenase complex.

The homo-24-meric dihydrolipoyl transacylase (E2) scaffold of the human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) contains the lipoyl-bearing domain (hbLBD), the subunit-binding domain (hbSBD) and the inner core domain that are linked to carry out E2 functions in substrate channeling and recognition. In this study, we employed NMR techniques to determine the structure of hbSBD and dynamics of several truncated constructs from the E2 component of the human BCKDC, including hbLBD (residues 1-84), hbSBD (residues 111-149), and a di-domain (hbDD) (residues 1-166) comprising hbLBD, hbSBD and the interdomain linker. The solution structure of hbSBD consists of two nearly parallel helices separated by a long loop, similar to the structures of the SBD isolated from other species, but it lacks the short 3(10) helix. The NMR results show that the structures of hbLBD and hbSBD in isolated forms are not altered by the presence of the interdomain linker in hbDD. The linker region is not entirely exposed to solvent, where amide resonances associated with approximately 50% of the residues are observable. However, the tethering of these two domains in hbDD significantly retards the overall rotational correlation times of hbLBD and hbSBD, changing from 5.54 ns and 5.73 ns in isolated forms to 8.37 ns and 8.85 ns in the linked hbDD, respectively. We conclude that the presence of the interdomain linker restricts the motional freedom of the hbSBD more significantly than hbLBD, and that the linker region likely exists as a soft rod rather than a flexible string in solution.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2006

Structural determinants for cross-talk between pyruvate dehydrogenase kinase 3 and lipoyl domain 2 of the human pyruvate dehydrogenase complex.

Pyruvate dehydrogenase kinase isoforms (PDK1-4) are the molecular switch that down-regulates activity of the human pyruvate dehydrogenase complex through reversible phosphorylation. We showed previously that binding of the lipoyl domain 2 (L2) of the pyruvate dehydrogenase complex to PDK3 induces a "cross-tail" conformation in PDK3, resulting in an opening of the active site cleft and the stimulation of kinase activity. In the present study, we report that alanine substitutions of Leu-140, Glu-170, and Glu-179 in L2 markedly reduce binding affinities of these L2 mutants for PDK3. Unlike wildtype L2, binding of these L2 mutants to PDK3 does not preferentially reduce the affinity of PDK3 for ADP over ATP. The inefficient removal of product inhibition associated with ADP accounts for the decreased stimulation of PDK3 activity by these L2 variants. Serial truncations of the PDK3 C-terminal tail region either impede or abolish the binding of wild-type L2 to the PDK3 mutants, resulting in the reduction or absence of L2-enhanced kinase activity. Alanine substitutions of residues Leu-27, Phe-32, Phe-35, and Phe-48 in the lipoyl-binding pocket of PDK3 similarly nullify L2 binding and L2-stimulated PDK3 activity. Our results indicate that the above residues in L2 and residues in the C-terminal region and the lipoyl-binding pocket of PDK3 are critical determinants for the cross-talk between L2 and PDK3, which up-regulates PDK3 activity.

Adenosine Diphosphate↗

PubMed2006

Effect of methamphetamine self-administration on tyrosine hydroxylase and dopamine transporter levels in mesolimbic and nigrostriatal dopamine pathways of the rat.

RATIONALE AND OBJECTIVES: Many studies have examined the effect of experimenter-delivered methamphetamine on the mesolimbic and nigrostriatal dopamine pathways. In contrast, little is known about the effect of methamphetamine self-administration on these neuronal pathways. We studied the effect of methamphetamine self-administration on two key regulators of dopamine transmission, tyrosine hydroxylase (TH), and dopamine transporter (DAT), in components of the mesolimbic and nigrostriatal dopamine pathways. METHODS: Rats self-administered methamphetamine (0.1 mg/kg per infusion, fixed-ratio-1 reinforcement schedule) or saline (control condition) for 9 h/day over 10 days. The brains of these rats were collected after 1 or 30 days of forced abstinence and the expression levels of TH and DAT were assayed by in situ, hybridization and western blot. RESULTS: TH mRNA and protein levels were increased in the ventral tegmental area (VTA, the cell body region of the mesolimbic dopamine system) and the substantia nigra pars compacta (SNC, the cell body region of the nigrostriatal dopamine system) after 1 day, but not 30 days, of forced abstinence from methamphetamine. In contrast, methamphetamine self-administration had no effect on TH protein levels in dopaminergic terminals located in the nucleus accumbens and caudate-putamen. In addition, methamphetamine self-administration had no effect on DAT mRNA levels in the VTA. CONCLUSIONS: Results suggest that extended daily access to self-administered methamphetamine results in a transient, short-lasting effect on mesolimbic and nigrostriatal dopamine neurons of the rat brain.

Animals↗

PubMed2006

Structural insight into interactions between dihydrolipoamide dehydrogenase (E3) and E3 binding protein of human pyruvate dehydrogenase complex.

The 9.5 MDa human pyruvate dehydrogenase complex (PDC) utilizes the specific dihydrolipoamide dehydrogenase (E3) binding protein (E3BP) to tether the essential E3 component to the 60-meric core of the complex. Here, we report crystal structures of the binding domain (E3BD) of human E3BP alone and in complex with human E3 at 1.6 angstroms and 2.2 angstroms, respectively. The latter structure shows that residues from E3BD contact E3 across its 2-fold axis, resulting in one E3BD binding site on the E3 homodimer. Negligible conformational changes occur in E3BD upon its high-affinity binding to E3. Modifications of E3BD residues at the center of the E3BD/E3 interface impede E3 binding far more severely than those of residues on the periphery, validating the "hot spot" paradigm for protein interactions. A cluster of disease-causing E3 mutations located near the center of the E3BD/E3 interface prevents the efficient recruitment of these E3 variants by E3BP into the PDC, leading to the dysfunction of the PDC catalytic machine.

Amino Acid Sequence↗

PubMed2006

A versatile conformational switch regulates reactivity in human branched-chain alpha-ketoacid dehydrogenase.

The dehydrogenase/decarboxylase (E1b) component of the 4 MD human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is a thiamin diphosphate (ThDP)-dependent enzyme. We have determined the crystal structures of E1b with ThDP bound intermediates after decarboxylation of alpha-ketoacids. We show that a key tyrosine residue in the E1b active site functions as a conformational switch to reduce the reactivity of the ThDP cofactor through interactions with its thiazolium ring. The intermediates do not assume the often-postulated enamine state, but likely a carbanion state. The carbanion presumably facilitates the second E1b-catalyzed reaction, involving the transfer of an acyl moiety from the intermediate to a lipoic acid prosthetic group in the transacylase (E2b) component of the BCKDC. The tyrosine switch further remodels an E1b loop region to promote E1b binding to E2b. Our results illustrate the versatility of the tyrosine switch in coordinating the catalytic events in E1b by modulating the reactivity of reaction intermediates.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2006

An expanded conformation of single-ring GroEL-GroES complex encapsulates an 86 kDa substrate.

Electron cryomicroscopy reveals an unprecedented conformation of the single-ring mutant of GroEL (SR398) bound to GroES in the presence of Mg-ATP. This conformation exhibits a considerable expansion of the folding cavity, with approximately 80% more volume than the X-ray structure of the equivalent cis cavity in the GroEL-GroES-(ADP)(7) complex. This expanded conformation can encapsulate an 86 kDa heterodimeric (alphabeta) assembly intermediate of mitochondrial branched-chain alpha-ketoacid dehydrogenase, the largest substrate ever observed to be cis encapsulated. The SR398-GroES-Mg-ATP complex is found to exist as a mixture of standard and expanded conformations, regardless of the absence or presence of the substrate. However, the presence of even a small substrate causes a pronounced bias toward the expanded conformation. Encapsulation of the large assembly intermediate is supported by a series of electron cryomicroscopy studies as well as the protection of both alpha and beta subunits of the substrate from tryptic digestion.

Adenosine Triphosphate↗

PubMed2006

Lessons from genetic disorders of branched-chain amino acid metabolism.

Genetic disorders of BCAA metabolism produce amino acidopathies and various forms of organic aciduria with severe clinical consequences. A metabolic block in the oxidative decarboxylation of BCAA caused by mutations in the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC) results in Maple Syrup Urine Disease (MSUD) or branched-chain ketoaciduria. There are presently five known clinical phenotypes for MSUD, i.e., classic, intermediate, intermittent, thiamin-responsive, and dihydrolipoamide dehydrogenase (E3)-deficient, based on severity of the disease, response to thiamin therapy, and the gene locus affected. Reduced glutamate, glutamine, and gamma-aminobutyrate concentrations induced by the accumulation of branched-chain alpha-ketoacids in the brain cortex of affected children and neonatal polled Hereford calves are considered the cause of MSUD encephalopathies. The long-term restriction of BCAA intake in diets and orthotopic liver transplantation have proven effective in controlling plasma BCAA levels and mitigating some of the above neurological manifestations. To date, approximately 100 mutations have been identified in four (branched-chain alpha-ketoacid decarboxylase/dehydrogenasealpha [E1alpha], E1beta, dihydrolipoyl transacylase [E2], and E3) of the six genes that encode the human BCKDC catalytic machine. We have documented a strong correlation between the presence of mutant E2 proteins and the thiamin-responsive MSUD phenotype. We show that the normal E1 component possesses residual decarboxylase activity, which is augmented by the binding to a mutant E2 protein in the presence of the E1 cofactor thiamin diphosphate. Our results provide a biochemical model for the effectiveness of thiamin therapy to thiamin-responsive MSUD patients.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2005

Crystal structure of human dihydrolipoamide dehydrogenase: NAD+/NADH binding and the structural basis of disease-causing mutations.

Human dihydrolipoamide dehydrogenase (hE3) is an enzymatic component common to the mitochondrial alpha-ketoacid dehydrogenase and glycine decarboxylase complexes. Mutations to this homodimeric flavoprotein cause the often-fatal human disease known as E3 deficiency. To catalyze the oxidation of dihydrolipoamide, hE3 uses two molecules: non-covalently bound FAD and a transiently bound substrate, NAD+. To address the catalytic mechanism of hE3 and the structural basis for E3 deficiency, the crystal structures of hE3 in the presence of NAD+ or NADH have been determined at resolutions of 2.5A and 2.1A, respectively. Although the overall fold of the enzyme is similar to that of yeast E3, these two structures differ at two loops that protrude from the proteins and at their FAD-binding sites. The structure of oxidized hE3 with NAD+ bound demonstrates that the nicotinamide moiety is not proximal to the FAD. When NADH is present, however, the nicotinamide base stacks directly on the isoalloxazine ring system of the FAD. This is the first time that this mechanistically requisite conformation of NAD+ or NADH has been observed in E3 from any species. Because E3 structures were previously available only from unicellular organisms, speculations regarding the molecular mechanisms of E3 deficiency were based on homology models. The current hE3 structures show directly that the disease-causing mutations occur at three locations in the human enzyme: the dimer interface, the active site, and the FAD and NAD(+)-binding sites. The mechanisms by which these mutations impede the function of hE3 are discussed.

Binding Sites↗

PubMed2005

Crystal structure of pyruvate dehydrogenase kinase 3 bound to lipoyl domain 2 of human pyruvate dehydrogenase complex.

The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3-L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity.

Adenosine Diphosphate↗

PubMed2004

Cross-talk between thiamin diphosphate binding and phosphorylation loop conformation in human branched-chain alpha-keto acid decarboxylase/dehydrogenase.

The decarboxylase/dehydrogenase (E1b) component of the 4-megadalton human branched-chain alpha-keto acid dehydrogenase (BCKD) metabolic machine is a thiamin diphosphate (ThDP)-dependent enzyme with a heterotetrameric cofactor-binding fold. The E1b component catalyzes the decarboxylation of alpha-keto acids and the subsequent reductive acylation of the lipoic acid-bearing domain (LBD) from the 24-meric transacylase (E2b) core. In the present study, we show that the binding of cofactor ThDP to the E1b active site induces a disorder-to-order transition of the conserved phosphorylation loop carrying the two phosphorylation sites Ser(292)-alpha and Ser(302)-alpha, as deduced from the 1.80-1.85 A apoE1b and holoE1b structures. The induced loop conformation is essential for the recognition of lipoylated LBD to initiate E1b-catalyzed reductive acylation. Alterations of invariant Arg(287)-alpha, Asp(295)-alpha, Tyr(300)-alpha, and Arg(301)-alpha that form a hydrogen-bonding network in the phosphorylation loop result in the disordering of the loop conformation as elucidated by limited proteolysis, accompanied by the impaired binding and diminished reductive acylation of lipoylated LBD. In contrast, k(cat) values for E1b-catalyzed decarboxylation of the alpha-keto acid are higher in these E1b mutants than in wild-type E1b, with higher K(m) values for the substrate in the mutants. ThDP binding that orders the loop prevents phosphorylation of E1b by the BCKD kinase and averts the inactivation of wild-type E1b, but not the above mutants, by this covalent modification. Our results establish that the cross-talk between the bound ThDP and the phosphorylation loop conformation serves as a feed-forward switch for multiple reaction steps in the BCKD metabolic machine.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2004

Structural and biochemical basis for novel mutations in homozygous Israeli maple syrup urine disease patients: a proposed mechanism for the thiamin-responsive phenotype.

Maple syrup urine disease (MSUD) results from mutations affecting different subunits of the mitochondrial branched-chain alpha-ketoacid dehydrogenase complex. In this study, we identified seven novel mutations in MSUD patients from Israel. These include C219W-alpha (TGC to TGG) in the E1alpha subunit; H156Y-beta (CAT to TAT), V69G-beta (GTT to GGT), IVS 9 del[-7:-4], and 1109 ins 8bp (exon 10) in the E1beta subunit; and H391R (CAC to CGC) and S133stop (TCA to TGA) affecting the E2 subunit of the branched-chain alpha-ketoacid dehydrogenase complex. Recombinant E1 proteins carrying the C219W-alpha or H156Y-beta mutation show no catalytic activity with defective subunit assembly and reduced binding affinity for cofactor thiamin diphosphate. The mutant E1 harboring the V69G-beta substitution cannot be expressed, suggesting aberrant folding caused by this mutation. These E1 mutations are ubiquitously associated with the classic phenotype in homozygous-affected patients. The H391R substitution in the E2 subunit abolishes the key catalytic residue that functions as a general base in the acyltransfer reaction, resulting in a completely inactive E2 component. However, wild-type E1 activity is enhanced by E1 binding to this full-length mutant E2 in vitro. We propose that the augmented E1 activity is responsible for robust thiamin responsiveness in homozygous patients carrying the H391R E2 mutation and that the presence of a full-length mutant E2 is diagnostic of this MSUD phenotype. The present results offer a structural and biochemical basis for these novel mutations and will facilitate DNA-based diagnosis for MSUD in the Israeli population.

Alleles↗

PubMed2004

Seeing GroEL at 6 A resolution by single particle electron cryomicroscopy.

We present a reconstruction of native GroEL by electron cryomicroscopy (cryo-EM) and single particle analysis at 6 A resolution. alpha helices are clearly visible and beta sheet density is also visible at this resolution. While the overall conformation of this structure is quite consistent with the published X-ray data, a measurable shift in the positions of three alpha helices in the intermediate domain is observed, not consistent with any of the 7 monomeric structures in the Protein Data Bank model (1OEL). In addition, there is evidence for slight rearrangement or flexibility in parts of the apical domain. The 6 A resolution cryo-EM GroEL structure clearly demonstrates the veracity and expanding scope of cryo-EM and the single particle reconstruction technique for macromolecular machines.

Chaperonin 60↗

PubMed2004

Molecular mechanism for regulation of the human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex by phosphorylation.

The human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is a 4 MDa macromolecular machine comprising three catalytic components (E1b, E2b, and E3), a kinase, and a phosphatase. The BCKDC overall activity is tightly regulated by phosphorylation in response to hormonal and dietary stimuli. We report that phosphorylation of Ser292-alpha in the E1b active site channel results in an order-to-disorder transition of the conserved phosphorylation loop carrying the phosphoryl serine. The conformational change is triggered by steric clashes of the phosphoryl group with invariant His291-alpha that serves as an indispensable anchor for the phosphorylation loop through bound thiamin diphosphate. Phosphorylation of Ser292-alpha does not severely impede the E1b-dependent decarboxylation of alpha-ketoacids. However, the disordered loop conformation prevents phosphorylated E1b from binding the E2b lipoyl-bearing domain, which effectively shuts off the E1b-catalyzed reductive acylation reaction and therefore completely inactivates BCKDC. This mechanism provides a paradigm for regulation of mitochondrial alpha-ketoacid dehydrogenase complexes by phosphorylation.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2003

Roles of His291-alpha and His146-beta' in the reductive acylation reaction catalyzed by human branched-chain alpha-ketoacid dehydrogenase: refined phosphorylation loop structure in the active site.

We report here that alterations of either His291-alpha or His146-beta' in the active site of human branched-chain alpha-ketoacid dehydrogenase (E1b) impede both the decarboxylation and the reductive acylation reactions catalyzed by E1b as well as the binding of cofactor thiamin diphosphate (ThDP). In a refined human E1b active-site structure, His291-alpha, which aligns with His407 in Escherichia coli pyruvate dehydrogenase and His263 in yeast transketolase, is on a largely ordered phosphorylation loop. The imidazole ring of His291-alpha in E1b coordinates to the terminal phosphate oxygen atoms of bound ThDP. The N3 atom of wild-type His146-beta', which can be protonated, binds a water molecule and points toward the aminopyrimidine ring of ThDP. Remarkably, the H291A-alpha mutation results in a complete order-to-disorder transition of the loop region, which precludes the binding of the substrate lipoyl-bearing domain to E1b. The H146A-beta' mutation, on the other hand, does not alter the loop structure, but nullifies the reductive acylation activity of E1b. Our results suggest that: 1) His291-alpha plays a structural rather than a catalytic role in the binding of cofactor ThDP and the lipoyl-bearing domain to E1b, and 2) His146-beta' is an essential catalytic residue, probably functioning as a proton donor in the reductive acylation of lipoamide on the lipoyl-bearing domain.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2002

Encapsulation of an 86-kDa assembly intermediate inside the cavities of GroEL and its single-ring variant SR1 by GroES.

We described previously that during the assembly of the alpha(2)beta(2) heterotetramer of human mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD), chaperonins GroEL/GroES interact with the kinetically trapped heterodimeric (alphabeta) intermediate to facilitate conversion of the latter to the native BCKD heterotetramer. Here, we show that the 86-kDa heterodimeric intermediate possesses a native-like conformation as judged by its binding to a fluorescent probe 1-anilino-8-naphthalenesulfonate. This large heterodimeric intermediate is accommodated as an entity inside cavities of GroEL and its single-ring variant SR1 and is encapsulated by GroES as indicated by the resistance of the heterodimer to tryptic digestion. The SR1-alphabeta-GroES complex is isolated as a stable single species by gel filtration in the presence of Mg-ATP. In contrast, an unfolded BCKD fusion protein of similar size, which also resides in the GroEL or SR1 cavity, is too large to be capped by GroES. The cis-capping mechanism is consistent with the high level of BCKD activity recovered with the GroEL-alphabeta complex, GroES, and Mg-ATP. The 86-kDa native-like heterodimeric intermediate in the BCKD assembly pathway represents the largest protein substrate known to fit inside the GroEL cis cavity underneath GroES, which significantly exceeds the current size limit of 57 kDa established for unfolded proteins.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2002

The C-terminal hinge region of lipoic acid-bearing domain of E2b is essential for domain interaction with branched-chain alpha-keto acid dehydrogenase kinase.

The branched-chain alpha-keto acid dehydrogenase (BCKD) kinase (abbreviated as BCK) down-regulates activity of the mammalian mitochondrial BCKD complex by reversible phosphorylation of the decarboxylase (E1b) component of the complex. The binding of BCK to the holotransacylase (E2b) core of the BCKD complex results in the stimulation of BCK activity. Here we show that the lipoylated lipoic acid-bearing domain (lip-LBD) (residues 1-84) of E2b alone does not interact with BCK. However, lip-LBD constructs containing various lengths of the C-terminal hinge region of LBD are able to bind to BCK as measured by a newly developed solubility-based binding assay. Isothermal titration calorimetry measurements produced a dissociation constant of 8.06 x 10(-6) m and binding enthalpy of -3.68 kcal/mol for the interaction of BCK with a construct containing lip-LBD and the Glu-Glu-Asp-Xaa-Xaa-Glu sequence of the C-terminal hinge region of LBD. These thermodynamic parameters are similar to those obtained for binding of BCK to a lipoylated di-domain construct, which harbors LBD, the entire hinge region, and the downstream subunit-binding domain of E2b. Our data establish that the C-terminal hinge region of LBD containing the above negatively charged residues is essential for the interaction between the lip-LBD construct and BCK.

Animals↗