Biomedical subjects

Liang Tong

Publications and source records attributed to Liang Tong.

At least 37 records · Page 2Linked to original sources

PubMed2005

Crystal structure of human Taspase1, a crucial protease regulating the function of MLL.

Taspase1 catalyzes the proteolytic processing of the mixed lineage leukemia (MLL) nuclear protein, which is required for maintaining Hox gene expression patterns. Chromosomal translocations of the MLL gene are associated with leukemia in infants. Taspase1, a threonine aspartase, is a member of the type 2 asparaginase family, but is the only protease in this family. We report here the crystal structures of human activated Taspase1 and its proenzyme, as well as the characterization of the effects of mutations in the active site region using a newly developed fluorogenic assay. The structure of Taspase1 has significant differences from other asparaginases, especially near the active site. Mutation of the catalytic nucleophile, Thr234, abolishes autocatalytic processing in cis but does not completely block proteolysis in trans. The structure unexpectedly showed the binding of a chloride ion in the active site, and our kinetic studies confirm that chlorides ions are inhibitors of this enzyme at physiologically relevant concentrations.

Amino Acid Sequence↗

PubMed2004

A mechanism for the potent inhibition of eukaryotic acetyl-coenzyme A carboxylase by soraphen A, a macrocyclic polyketide natural product.

Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism. Soraphen A, a macrocyclic polyketide natural product, is a nanomolar inhibitor against the biotin carboxylase (BC) domain of human, yeast, and other eukaryotic ACCs. Here we report the crystal structures of the yeast BC domain, alone and in complex with soraphen A. Soraphen has extensive interactions with an allosteric site, about 25 A from the active site. The specificity of soraphen is explained by large structural differences between the eukaryotic and prokaryotic BC in its binding site, confirmed by our studies on the effects of single-site mutations in this binding site. Unexpectedly, our structures suggest that soraphen may bind in the BC dimer interface and inhibit the BC activity by disrupting the oligomerization of this domain. Observations from native gel electrophoresis confirm this structural insight. The structural information provides a foundation for structure-based design of new inhibitors against these enzymes.

Acetyl-CoA Carboxylase↗

PubMed2004

Crystal structure of the PH-BEACH domains of human LRBA/BGL.

The beige and Chediak-Higashi syndrome (BEACH) domain defines a large family of eukaryotic proteins that have diverse cellular functions in vesicle trafficking, membrane dynamics, and receptor signaling. The domain is the only module that is highly conserved among all of these proteins, but the exact functions of this domain and the molecular basis for its actions are currently unknown. Our previous studies showed that the BEACH domain is preceded by a novel, weakly conserved pleckstrin homology (PH) domain. We report here the crystal structure at 2.4 A resolution of the PH-BEACH domain of human LRBA/BGL. The PH domain has the same backbone fold as canonical PH domains, despite sharing no sequence homology with them. However, our binding assays demonstrate that the PH domain in the BEACH proteins cannot bind phospholipids. The BEACH domain contains a core of several partially extended peptide segments that is flanked by helices on both sides. The structure suggests intimate association between the PH and the BEACH domains, and surface plasmon resonance studies confirm that the two domains of the protein FAN have high affinity for each other, with a K(d) of 120 nM.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Structure and function of the phenazine biosynthetic protein PhzF from Pseudomonas fluorescens.

Phenazines produced by Pseudomonas and Streptomyces spp. are heterocyclic nitrogen-containing metabolites with antibiotic, antitumor, and antiparasitic activity. The antibiotic properties of pyocyanin, produced by Pseudomonas aeruginosa, were recognized in the 1890s, although this blue phenazine is now known to be a virulence factor in human disease. Despite their biological significance, the biosynthesis of phenazines is not fully understood. Here we present structural and functional studies of PhzF, an enzyme essential for phenazine synthesis in Pseudomonas spp. PhzF shares topology with diaminopimelate epimerase DapF but lacks the same catalytic residues. The structure of PhzF in complex with its substrate, trans-2,3-dihydro-3-hydroxyanthranilic acid, suggests that it is an isomerase using the conserved glutamate E45 to abstract a proton from C3 of the substrate. The proton is returned to C1 of the substrate after rearrangement of the double-bond system, yielding an enol that converts to the corresponding ketone. PhzF is a dimer that may be bifunctional, providing a shielded cavity for ketone dimerization via double Schiff-base formation to produce the phenazine scaffold. Our proposed mechanism is supported by mass and NMR spectroscopy. The results are discussed in the context of related structures and protein sequences of unknown biochemical function.

Bacterial Proteins↗

PubMed2004

Crystal structure of mouse carnitine octanoyltransferase and molecular determinants of substrate selectivity.

Carnitine acyltransferases have crucial functions in fatty acid metabolism. Members of this enzyme family show distinctive substrate preferences for short-, medium- or long-chain fatty acids. The molecular mechanism for this substrate selectivity is not clear as so far only the structure of carnitine acetyltransferase has been determined. To further our understanding of these important enzymes, we report here the crystal structures at up to 2.0-A resolution of mouse carnitine octanoyltransferase alone and in complex with the substrate octanoylcarnitine. The structures reveal significant differences in the acyl group binding pocket between carnitine octanoyltransferase and carnitine acetyltransferase. Amino acid substitutions and structural changes produce a larger hydrophobic pocket that binds the octanoyl group in an extended conformation. Mutation of a single residue (Gly-553) in this pocket can change the substrate preference between short- and medium-chain acyl groups. The side chains of Cys-323 and Met-335 at the bottom of this pocket assume dual conformations in the substrate complex, and mutagenesis studies suggest that the Met-335 residue is important for catalysis.

Amino Acid Sequence↗

PubMed2004

Dual functional roles of ATP in the human mitochondrial malic enzyme.

Human mitochondrial malic enzyme is a regulatory enzyme with ATP as an inhibitor. Structural studies reveal that the enzyme has two ATP-binding sites, one at the NAD(+)-binding site in the active center and the other at the exo site in the tetramer interface. Inhibition of the enzyme activity is due to the competition between ATP and NAD(+) for the nucleotide-binding site at the active center with an inhibition constant of 81 microM. Binding of the ATP molecule at the exo site, on the other hand, is important for the maintenance of the quaternary structural integrity. The enzyme exists in solution at neutral pH and at equilibrium of the dimer and tetramer with a dissociation constant (K(TD)) of 0.67 microM. ATP, at a physiological concentration, shifts the equilibrium toward tetramer and decreases the K(TD) by many orders of magnitude. Mutation of a single residue Arg542 at the tetrameric interfacial exo site resulted in dimeric mutants. ATP thus has dual functional roles in the mitochondrial malic enzyme.

Adenosine Triphosphate↗

PubMed2004

Structural basis of the alpha1-beta subunit interaction of voltage-gated Ca2+ channels.

High-voltage-activated Ca2+ channels are essential for diverse biological processes. They are composed of four or five subunits, including alpha1, alpha2-delta, beta and gamma (ref. 1). Their expression and function are critically dependent on the beta-subunit, which transports alpha1 to the surface membrane and regulates diverse channel properties. It is believed that the beta-subunit interacts with alpha1 primarily through the beta-interaction domain (BID), which binds directly to the alpha-interaction domain (AID) of alpha1; however, the molecular mechanism of the alpha1-beta interaction is largely unclear. Here we report the crystal structures of the conserved core region of beta3, alone and in complex with AID, and of beta4 alone. The structures show that the beta-subunit core contains two interacting domains: a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain. The AID binds to a hydrophobic groove in the GK domain through extensive interactions, conferring extremely high affinity between alpha1 and beta-subunits. The BID is essential both for the structural integrity of and for bridging the SH3 and GK domains, but it does not participate directly in binding alpha1. The presence of multiple protein-interacting modules in the beta-subunit opens a new dimension to its function as a multi-functional protein.

Amino Acid Sequence↗

PubMed2004

Structural and biochemical studies of the substrate selectivity of carnitine acetyltransferase.

Carnitine acyltransferases catalyze the exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids, and are attractive targets for drug discovery against diabetes and obesity. These enzymes are classified based on their substrate selectivity for short-chain, medium-chain, or long-chain fatty acids. Structural information on carnitine acetyltransferase suggests that residues Met-564 and Phe-565 may be important determinants of substrate selectivity with the side chain of Met-564 located in the putative binding pocket for acyl groups. Both residues are replaced by glycine in carnitine palmitoyltransferases. To assess the functional relevance of this structural observation, we have replaced these two residues with small amino acids by mutagenesis, characterized the substrate preference of the mutants, and determined the crystal structures of two of these mutants. Kinetic studies confirm that the M564G or M564A mutation is sufficient to increase the activity of the enzyme toward medium-chain substrates with hexanoyl-CoA being the preferred substrate for the M564G mutant. The crystal structures of the M564G mutant, both alone and in complex with carnitine, reveal a deep binding pocket that can accommodate the larger acyl group. We have determined the crystal structure of the F565A mutant in a ternary complex with both the carnitine and CoA substrates at a 1.8-A resolution. The F565A mutation has minor effects on the structure or the substrate preference of the enzyme.

Acyl Coenzyme A↗

PubMed2004

Crystal structure of the Toll/interleukin-1 receptor domain of human IL-1RAPL.

The Toll/interleukin-1 receptor (TIR) domain is conserved in the intracellular regions of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) as well as in several cytoplasmic adapter molecules. This domain has crucial roles in signal transduction by these receptors for host immune response. Here we report the crystal structure at 2.3-A resolution of the TIR domain of human IL-1RAPL, the first structure of a TIR domain of the IL-1R superfamily. There are large structural differences between this TIR domain and that of TLR1 and TLR2. Helix alphaD in IL-1RAPL is almost perpendicular to its equivalent in TLR1 or TLR2. The BB loop contains a hydrogen bond unique to IL-1RAPL between Thr residues at the 8th and 10th positions. The structural and sequence diversity among these domains may be important for specificity in the signal transduction by these receptors. A dimer of the TIR domain of IL-1RAPL is observed in the crystal, although this domain is monomeric in solution. Residues in the dimer interface are mostly unique to IL-1RAPL, which is consistent with the distinct functional roles of this receptor. Our functional studies show IL-1RAPL can activate JNK but not the ERK or the p38 MAP kinases, whereas its close homolog, TIGIRR, cannot activate JNK. Deletion mutagenesis studies show that the activation of JNK by IL-1RAPL does not depend on the integrity of its TIR domain, suggesting a distinct mechanism of signaling through this receptor.

Amino Acid Sequence↗

PubMed2004

Molecular basis for the inhibition of the carboxyltransferase domain of acetyl-coenzyme-A carboxylase by haloxyfop and diclofop.

Acetyl-CoA carboxylases (ACCs) are crucial for the metabolism of fatty acids, making these enzymes important targets for the development of therapeutics against obesity, diabetes, and other diseases. The carboxyltransferase (CT) domain of ACC is the site of action of commercial herbicides, such as haloxyfop, diclofop, and sethoxydim. We have determined the crystal structures at up to 2.5-A resolution of the CT domain of yeast ACC in complex with the herbicide haloxyfop or diclofop. The inhibitors are bound in the active site, at the interface of the dimer of the CT domain. Unexpectedly, inhibitor binding requires large conformational changes for several residues in this interface, which create a highly conserved hydrophobic pocket that extends deeply into the core of the dimer. Two residues that affect herbicide sensitivity are located in this binding site, and mutation of these residues disrupts the structure of the domain. Other residues in the binding site are strictly conserved among the CT domains.

Acetyl-CoA Carboxylase↗

PubMed2004

Crystal structure of yeast acetyl-coenzyme A synthetase in complex with AMP.

Acetyl-coenzyme A synthetase (ACS) belongs to the family of AMP-forming enzymes that also includes acyl-CoA synthetases, firefly luciferase, and nonribosomal peptide synthetases. ACS catalyzes the two-step activation of acetate to acetyl-CoA: formation of an acetyl-AMP intermediate from acetate and ATP and the transfer of the acetyl group to CoA. In mammals, the acetyl-CoA product is used for biosynthesis of long chain fatty acids as well as energy production. We have determined the crystal structure of yeast ACS in a binary complex with AMP at 2.3 A resolution. The structure contains a large, N-terminal domain and a small, C-terminal domain. AMP is bound at the interface between the two domains. This structure represents a new conformation for the ACS enzyme, which may be competent for catalyzing the first step of the reaction. A Lys residue that is critical for this step is located in the active site. A rotation of 140 degrees in the small domain is needed for the binding of CoA and the catalysis of the second step. In contrast to the monomeric bacterial enzyme, yeast ACS is a stable trimer.

Acetate-CoA Ligase↗

PubMed2004

Metal-Induced reversible structural interconversion of human mitochondrial NAD(P)+-dependent malic enzyme.

Human mitochondrial NAD(P)+-dependent malic enzyme was strongly inhibited by Lu3+. The X-ray crystal structures indicated a structural change between the metal-free and Lu3+-containing enzymes (Yang Z, Batra R, Floyd DL, Hung HC, Chang GG, Tong L. Biochem Biophys Res Commun 2000;274:440-444). We characterized the reversible slow-binding mechanism and the structural interconversion between Mn2+- and Lu3+-containing human mitochondrial malic enzymes. When Lu3+ was added, the activity of the human enzyme showed a downward curve over time, similar to that of the pigeon enzyme. The rate of the transformation (k(obs)) from the initial rate to the steady-state rate increased hyperbolically with the concentration of Lu3+, suggesting the involvement of an isomerization step. Lu3+ had a much higher affinity for the isomerized form (K*(i,Lu (app)) = 4.8 microM) than that of the native form (K(i,Lu (app)) = 148 microM). When an excess of Mn2+ was added to the Lu3+-inhibited enzyme, assays of the kinetic activity showed an upward trend, indicating reactivation. This result also indicated that the reactivation was a slow process. Fluorescence quenching experiments confirmed that the Lu3+-induced isomerization was completely reversible. The dynamic quenching constants for the metal-free, Mn2+-containing, and Lu3+-containing enzyme were 3.08, 3.07, and 3.8 M(-1), respectively. When the Lu3+-containing enzyme was treated with excess Mn2+, the dynamic quenching constant returned to the original value (3.09 M(-1)). These results indicated that binding of Mn2+ did not induce any conformational change in the enzyme. The open form transformed to the closed form only after substrate binding. Lu3+, on the other hand, transformed the open form into a catalytically inactive form. Excess Mn2+ could replace Lu3+ in the metal binding site and convert the inactive form back into the open form. This reversible process was slow in both directions because of the same but opposite structural change involved.

Binding Sites↗

PubMed2004

Characterization of the monomer-dimer equilibrium of human cytomegalovirus protease by kinetic methods.

Herpesviruses encode a serine protease that is essential for the maturation of infectious virions. This protease has a unique polypeptide backbone fold and contains a novel Ser-His-His catalytic triad. It exists in a monomer-dimer equilibrium in solution, but only the dimer form of the enzyme is catalytically active. The stability of this dimer is affected by the presence of anti-chaotropic agents. Most of the reported Kd values for this dimer (between 0.6 and 6 microM) are inconsistent with the fact that the protease is routinely assayed at 20-50 nM concentrations, as only monomeric species would be expected with such Kd values. We have characterized the monomer-dimer equilibrium of HCMV protease using a new method, which observes the exchange between dimers of the wild-type enzyme and the active-site Ser132Ala mutant in a titration experiment. The Kd of the dimer was determined to be 8 microM and 31 nM in the absence or presence of anti-chaotropic agents (10% glycerol and 0.5 M Na2SO4), respectively. Detailed kinetic analysis also showed that, in addition to the 260-fold stabilization of the dimer, the anti-chaotropic agents produced a 7-fold enhancement in the catalytic activity of the dimer.

Buffers↗

PubMed2004

A novel NAD-binding protein revealed by the crystal structure of 2,3-diketo-L-gulonate reductase (YiaK).

Escherichia coli YiaK catalyzes the reduction of 2,3-diketo-L-gulonate in the presence of NADH. It belongs to a large family of oxidoreductases that is conserved in archaea, bacteria, and eukaryotes but shows no sequence homology to other proteins. We report here the crystal structures at up to 2.0-A resolution of YiaK alone and in complex with NAD-tartrate. YiaK has a new polypeptide backbone fold and a novel mode of recognizing the NAD cofactor. In addition, NAD is bound in an unusual conformation, at the interface of a dimer of the enzyme. The crystallographic analysis unexpectedly revealed the binding of tartrate in the active site. Enzyme kinetics studies confirm that tartrate and the related D-malate are inhibitors of YiaK. In contrast to most other enzymes where substrate binding produces a more closed conformation, the binding of NAD-tartrate to YiaK produces a more open active site. The free enzyme conformation is incompatible with NAD binding. His(44) is likely the catalytic residue of the enzyme.

Amino Acid Sequence↗

PubMed2004

Novel homozygous p.E64D mutation in DJ1 in early onset Parkinson disease (PARK7).

Mutations in the parkin gene have been identified as a common cause of autosomal recessive inherited Parkinson disease (PD) associated with early disease manifestation. However, based on linkage data, mutations in other genes contribute to the genetic heterogeneity of early-onset PD (EOPD). Recently, two mutations in the DJ1 gene were described as a second cause of autosomal recessive EOPD (PARK7). Analyzing the PARK7/DJ1 gene in 104 EOPD patients, we identified a third mutation, c.192G>C (p.E64D), associated with EOPD in a patient of Turkish ancestry and characterized the functional significance of this amino acid substitution. In the patient, a substantial reduction of dopamine uptake transporter (DAT) binding was found in the striatum using [(18)F]FP-CIT and PET, indicating a serious loss of presynaptic dopaminergic afferents. His sister, homozygous for E64D, was clinically unaffected but showed reduced dopamine uptake when compared with a clinically unaffected brother, who is heterozygous for E64D. We demonstrate by crystallography that the E64D mutation does not alter the structure of the DJ1 protein, however we observe a tendency towards decreased levels of the mutant protein when overexpressed in HEK293 or COS7 cells. Using immunocytochemistry in contrast to the homogenous nuclear and cytoplasmic staining in HEK293 cells overexpressing wild-type DJ1, about 5% of the cells expressing E64D and up to 80% of the cells expressing the recently described L166P mutation displayed a predominant nuclear localization of the mutant DJ1 protein.

Adult↗

PubMed2004

Crystal structure of the carboxyltransferase domain of acetyl-coenzyme A carboxylase in complex with CP-640186.

Acetyl-coenzyme A carboxylases (ACCs) are important targets for the development of therapeutic agents against obesity, diabetes, and other diseases. CP-640186 is a potent inhibitor of mammalian ACCs and can reduce body weight and improve insulin sensitivity in test animals. It is believed to target the carboxyltransferase (CT) domain of these enzymes. Here we report the crystal structure of the yeast CT domain in complex with CP-640186. The inhibitor is bound in the active site at the interface of a dimer of the CT domain. CP-640186 has tight interactions with the putative biotin binding site in the CT domain and demonstrates a distinct mode of inhibiting the CT activity as compared to the herbicides that inhibit plant ACCs. The affinity of inhibitors for the CT domain has been assessed using kinetic and fluorescence anisotropy binding studies. The structural information identifies three regions for drug binding in the active site of CT.

Acetyl-CoA Carboxylase↗

PubMed2004

Structure and function of carnitine acyltransferases.

Carnitine acyltransferases catalyze the exchange of acyl groups between carnitine and coenzyme A (CoA). These enzymes include carnitine acetyltransferase (CrAT), carnitine octanoyltransferase (CrOT), and carnitine palmitoyltransferases (CPTs). CPT-I and CPT-II are crucial for the beta-oxidation of long-chain fatty acids in the mitochondria by enabling their transport across the mitochondrial membrane. The activity of CPT-I is inhibited by malonyl-CoA, a crucial regulatory mechanism for fatty acid oxidation. Mutation or dysregulation of the CPT enzymes has been linked to many serious, even fatal human diseases, and these enzymes are promising targets for the development of therapeutic agents against type 2 diabetes and obesity. We have determined the crystal structures of murine CrAT, alone and in complex with its substrate carnitine or CoA. The structure contains two domains. Surprisingly, these two domains share the same backbone fold, which is also similar to that of chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. The active site is located at the interface between the two domains, in a tunnel that extends through the center of the enzyme. Carnitine and CoA are bound in this tunnel, on opposite sides of the catalytic His343 residue. The structural information provides a molecular basis for understanding the catalysis by carnitine acyltransferases and for designing their inhibitors. In addition, our structural information suggests that the substrate carnitine may assist the catalysis by stabilizing the oxyanion in the reaction intermediate.

Acetyltransferases↗

PubMed2003

Structure and function of malic enzymes, a new class of oxidative decarboxylases.

Malic enzyme is a tetrameric protein with double dimer structure in which the dimer interface is more intimately contacted than the tetramer interface. Each monomeric unit of the enzyme is composed of four structural domains, which show a different folding topology from those of the other oxidative decarboxylases. The active center is located at the interface between domains B and C. For human mitochondrial malic enzyme, there is an exo nucleotide-binding site for the inhibitor ATP and an allosteric site for the activator fumarate, located at the tetramer and dimer interfaces, respectively. Crystal structures of the enzyme in various complexed forms indicate that the enzyme may exist in equilibrium among two open and two closed forms. Interconversion among these forms involves rigid-body movements of the four structural domains. Substrate binding at the active site shifts the open form to the closed form that represents an active site closure. Fumarate binding at the allosteric site induces the interconversion between forms I and II, which is mediated by the movements of domains A and D. Structures of malic enzyme from different sources are compared with an emphasis on the differences and their implications to structure-function relationships. The binding modes of the substrate, product, cofactors, and transition-state analogue at the active site, as well as ATP and fumarate at the exo site and allosteric site, respectively, provide a clear account for the catalytic mechanism, nucleotide specificities, allosteric regulation, and functional roles of the quaternary structure. The proposed catalytic mechanism involves tyrosine-112 and lysine-183 as the general acid and base, respectively. In addition, a divalent metal ion (Mn(2+) or Mg(2+)) is essential in helping the catalysis. Binding of the metal ion also plays an important role in stabilizing the quaternary structural integrity of the enzyme.

Amino Acid Sequence↗