Jacinta L. Chuang

Crystal structure of human branched-chain α-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease

Abstract Background: Mutations in components of the extraordinarily large α-ketoacid dehydrogenase multienzyme complexes can lead to serious and often fatal disorders in humans, including maple syrup urine disease (MSUD). In order to obtain insight into the effect of mutations observed in MSUD patients, we determined the crystal structure of branched-chain α-ketoacid dehydrogenase (E1), the 170 kDa α 2 β 2 heterotetrameric E1b component of the branched-chain α-ketoacid dehydrogenase multienzyme complex. Results: The 2.7 A resolution crystal structure of human E1b revealed essentially the full α and β polypeptide chains of the tightly packed heterotetramer. The position of two important potassium (K + ) ions was determined. One of these ions assists a loop that is close to the cofactor to adopt the proper conformation. The second is located in the β subunit near the interface with the small C-terminal domain of the α subunit. The known MSUD mutations affect the functioning of E1b by interfering with the cofactor and K + sites, the packing of hydrophobic cores, and the precise arrangement of residues at or near several subunit interfaces. The Tyr→Asn mutation at position 393-α occurs very frequently in the US population of Mennonites and is located in a unique extension of the human E1b α subunit, contacting the β′ subunit. Conclusions: Essentially all MSUD mutations in human E1b can be explained on the basis of the structure, with the severity of the mutations for the stability and function of the protein correlating well with the severity of the disease for the patients. The suggestion is made that small molecules with high affinity for human E1b might alleviate effects of some of the milder forms of MSUD.

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.

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.

Structure of rat BCKD kinase: Nucleotide-induced domain communication in a mitochondrial protein kinase

Mitochondrial protein kinases (mPKs) are molecular switches that down-regulate the oxidation of branched-chain α-ketoacids and pyruvate. Elevated levels of these metabolites are implicated in disease states such as insulin-resistant Type II diabetes, branched-chain ketoaciduria, and primary lactic acidosis. We report a three-dimensional structure of a member of the mPK family, rat branched-chain α-ketoacid dehydrogenase kinase (BCK). BCK features a characteristic nucleotide-binding domain and a four-helix bundle domain. These two domains are reminiscent of modules found in protein histidine kinases (PHKs), which are involved in two-component signal transduction systems. Unlike PHKs, BCK dimerizes through direct interaction of two opposing nucleotide-binding domains. Nucleotide binding to BCK is uniquely mediated by both potassium and magnesium. Binding of ATP induces disorder–order transitions in a loop region at the nucleotide-binding site. These structural changes lead to the formation of a quadruple aromatic stack in the interface between the nucleotide-binding domain and the four-helix bundle domain, where they induce a movement of the top portion of two helices. Phosphotransfer induces further ordering of the loop region, effectively trapping the reaction product ADP, which explains product inhibition in mPKs. The BCK structure is a prototype for all mPKs and will provide a framework for structure-assisted inhibitor design for this family of kinases.

Structure of rat BCKD kinase

Mitochondrial protein kinases (mPKs) are molecular switches that down-regulate the oxidation of branched-chain α-ketoacids and pyruvate. Elevated levels of these metabolites are implicated in disease states such as insulin-resistant Type II diabetes, branched-chain ketoaciduria, and primary lactic acidosis. We report a three-dimensional structure of a member of the mPK family, rat branched-chain α-ketoacid dehydrogenase kinase (BCK). BCK features a characteristic nucleotide-binding domain and a four-helix bundle domain. These two domains are reminiscent of modules found in protein histidine kinases (PHKs), which are involved in two-component signal transduction systems. Unlike PHKs, BCK dimerizes through direct interaction of two opposing nucleotide-binding domains. Nucleotide binding to BCK is uniquely mediated by both potassium and magnesium. Binding of ATP induces disorder–order transitions in a loop region at the nucleotide-binding site. These structural changes lead to the formation of a quadruple aromatic stack in the interface between the nucleotide-binding domain and the four-helix bundle domain, where they induce a movement of the top portion of two helices. Phosphotransfer induces further ordering of the loop region, effectively trapping the reaction product ADP, which explains product inhibition in mPKs. The BCK structure is a prototype for all mPKs and will provide a framework for structure-assisted inhibitor design for this family of kinases.

Structural basis for inactivation of the human pyruvate dehydrogenase complex by phosphorylation: role of disordered phosphorylation loops.

We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-alpha (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-alpha prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-alpha, which nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p, negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine.

Pyruvate Dehydrogenase Kinase-4 Structures Reveal a Metastable Open Conformation Fostering Robust Core-free Basal Activity

Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1–4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-Å crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, compared with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp394–Trp395) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core.

Impaired Assembly of E1 Decarboxylase of the Branched-chain α-Ketoacid Dehydrogenase Complex in Type IA Maple Syrup Urine Disease

The E1 decarboxylase component of the human branched-chain ketoacid dehydrogenase complex comprises two E1α (45.5 kDa) and two E1β (37.5 kDa) subunits forming an α2β2 tetramer. In patients with type IA maple syrup urine disease, the E1α subunit is affected, resulting in the loss of E1 and branched-chain ketoacid dehydrogenase catalytic activities. To study the effect of human E1α missense mutations on E1 subunit assembly, we have developed a pulse-chase labeling protocol based on efficient expression and assembly of human (His)6-E1α and untagged E1β subunits inEscherichia coli in the presence of overexpressed chaperonins GroEL and GroES. Assembly of the two35S-labeled E1 subunits was indicated by their co-extraction with Ni2+-nitrilotriacetic acid resin. The nine E1α maple syrup urine disease mutants studied showed aberrant kinetics of assembly with normal E1β in the 2-h chase compared with the wild type and can be classified into four categories of normal (N222S-α and R220W-α), moderately slow (G245R-α), slow (G204S-α, A240P-α, F364C-α, Y368C-α, and Y393N-α), and no (T265R-α) assembly. Prolonged induction in E. coli grown in the YTGK medium or lowering of induction temperature from 37 to 28 °C (in the case of T265R-α), however, resulted in the production of mutant E1 proteins. Separation of purified E1 proteins by sucrose density gradient centrifugation showed that the wild-type E1 existed entirely as α2β2 tetramers. In contrast, a subset of E1α missense mutations caused the occurrence of exclusive αβ dimers (Y393N-α and F364C-α) or of both α2β2 tetramers and lower molecular weight species (Y368C-α and T265R-α). Thermal denaturation at 50 °C indicated that mutant E1 proteins aggregated more rapidly than wild type (rate constant, 0.19 min−1), with the T265R-α mutant E1 most severely affected (rate constant, 4.45 min−1). The results establish that the human E1α mutations in the putative thiamine pyrophosphate-binding pocket that are studied, with the exception of G204S-α, have no effect on E1 subunit assembly. The T265R-α mutation adversely impacts both E1α folding and subunit interactions. The mutations involving the C-terminal aromatic residues impede both the kinetics of subunit assembly and the formation of the native α2β2 structure.

Subunit and Catalytic Component Stoichiometries of an in Vitro Reconstituted Human Pyruvate Dehydrogenase Complex

The human pyruvate dehydrogenase complex (PDC) is a 9.5-megadalton catalytic machine that employs three catalytic components, i.e. pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), to carry out the oxidative decarboxylation of pyruvate. The human PDC is organized around a 60-meric dodecahedral core comprising the C-terminal domains of E2p and a noncatalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the PDC. A central issue concerning the PDC structure is the subunit stoichiometry of the E2p/E3BP core; recent studies have suggested that the core is composed of 48 copies of E2p and 12 copies of E3BP. Here, using an in vitro reconstituted PDC, we provide densitometry, isothermal titration calorimetry, and analytical ultracentrifugation evidence that there are 40 copies of E2p and 20 copies of E3BP in the E2p/E3BP core. Reconstitution with saturating concentrations of E1p and E3 demonstrated 40 copies of E1p heterotetramers and 20 copies of E3 dimers associated with the E2p/E3BP core. To corroborate the 40/20 model of this core, the stoichiometries of E3 and E1p binding to their respective binding domains were reexamined. In these binding studies, the stoichiometries were found to be 1:1, supporting the 40/20 model of the core. The overall maximal stoichiometry of this in vitro assembled PDC for E2p:E3BP:E1p:E3 is 40:20:40:20. These findings contrast a previous report that implicated that two E3-binding domains of E3BP bind simultaneously to a single E3 dimer (Smolle, M., Prior, A. E., Brown, A. E., Cooper, A., Byron, O., and Lindsay, J. G. (2006) J. Biol. Chem. 281, 19772–19780).

Structure-guided Development of Specific Pyruvate Dehydrogenase Kinase Inhibitors Targeting the ATP-binding Pocket

Background: Up-regulated pyruvate dehydrogenase kinase isoforms (PDKs) are associated with impaired glucose homeostasis in diabetes. Results: Novel PDK inhibitors were developed using structure-based design, which improves glucose tolerance with reduced hepatic steatosis in diet-induced obese mice. Conclusion: Obesity phenotypes are effectively treated by chemical intervention with PDK inhibitors. Significance: PDKs are potential drug targets for obesity and type 2 diabetes. Pyruvate dehydrogenase kinase isoforms (PDKs 1–4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 μm for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes.