Biomedical subjects
Publications and source records attributed to Liang Tong.
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Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid beta-oxidation. Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia. We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E. Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations. To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase. This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant). Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class. They are located more than 20 A away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex. By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category. They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms. This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders.
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We report the crystal structure at 1.8-A resolution of human DJ-1, which has been linked to early onset Parkinson's disease. The monomer of DJ-1 contains the alpha/beta-fold that is conserved among members of the DJ-1/ThiJ/PfpI superfamily. However, the structure also contains an extra helix at the C terminus, which mediates a novel mode of dimerization for the DJ-1 proteins. A putative active site has been identified near the dimer interface, and the residues Cys-106, His-126, and Glu-18 may play important roles in the catalysis by this protein. Studies with the disease-causing L166P mutant suggest that the mutation has disrupted the C-terminal region and the dimerization of the protein. The DJ-1 proteins may function only as dimers. The Lys to Arg mutation at residue 130, the site of sumoylation of DJ-1, has minimal impact on the structure of the protein.
Acetyl-coenzyme A carboxylases (ACCs) are required for the biosynthesis and oxidation of long-chain fatty acids. They are targets for therapeutics against obesity and diabetes, and several herbicides function by inhibiting their carboxyltransferase (CT) domain. We determined the crystal structure of the free enzyme and the coenzyme A complex of yeast CT at 2.7 angstrom resolution and found that it comprises two domains, both belonging to the crotonase/ClpP superfamily. The active site is at the interface of a dimer. Mutagenesis and kinetic studies reveal the functional roles of conserved residues here. The herbicides target the active site of CT, providing a lead for inhibitor development against human ACCs.
Herpesvirus protease is required for the life cycle of the virus and is an attractive target for the design and development of new anti-herpes agents. The protease belongs to a new class of serine proteases, with a novel backbone fold and a unique Ser-His-His catalytic triad. Here we report the crystal structures of human cytomegalovirus protease in complex with two peptidomimetic inhibitors. The structures reveal a new hydrogen-bonding interaction between the main chain carbonyl of the P(5) residue and the main chain amide of amino acid 137 of the protease, which is important for the binding affinity of the inhibitor. Conformational flexibility was observed in the S(3) pocket of the enzyme, and this is supported by our characterization of several mutants in this pocket. One of the structures is at 2.5 A resolution, allowing us for the first time to locate ordered solvent molecules in the inhibitor complex. The presence of two solvent molecules in the active site may have implications for the design of new inhibitors against this enzyme. Favorable and stereospecific interactions have been established in the S(1)' pocket for one of these inhibitors.
Carnitine acyltransferases have crucial roles in the transport of fatty acids for beta-oxidation. Dysregulation of these enzymes can lead to serious diseases in humans, and they are targets for therapeutic development against diabetes. We report the crystal structures of murine carnitine acetyltransferase (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. Carnitine and CoA are bound in deep channels in the enzyme, 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. Specifically, our structural information suggests that the substrate carnitine may assist the catalysis by stabilizing the oxyanion in the reaction intermediate.
Malic enzymes catalyze the oxidative decarboxylation of L-malate to pyruvate and CO(2) with the reduction of the NAD(P)(+) cofactor in the presence of divalent cations. We report the crystal structures at up to 2.1 A resolution of human mitochondrial NAD(P)(+)-dependent malic enzyme in different pentary complexes with the natural substrate malate or pyruvate, the dinucleotide cofactor NAD(+) or NADH, the divalent cation Mn(2+), and the allosteric activator fumarate. Malate is bound deep in the active site, providing two ligands for the cation, and its C4 carboxylate group is out of plane with the C1-C2-C3 atoms, facilitating decarboxylation. The divalent cation is positioned optimally to catalyze the entire reaction. Lys183 is the general base for the oxidation step, extracting the proton from the C2 hydroxyl of malate. Tyr112-Lys183 functions as the general acid-base pair to catalyze the tautomerization of the enolpyruvate product from decarboxylation to pyruvate.
In enzyme catalysis, where exquisitely positioned functionality is the sine qua non, atomic coordinates for a Michaelis complex can provide powerful insights into activation of the substrate. We focus here on the initial proton transfer of the isomerization reaction catalyzed by triosephosphate isomerase and present the crystal structure of its Michaelis complex with the substrate dihydroxyacetone phosphate at near-atomic resolution. The active site is highly compact, with unusually short and bifurcated hydrogen bonds for both catalytic Glu-165 and His-95 residues. The carboxylate oxygen of the catalytic base Glu-165 is positioned in an unprecedented close interaction with the ketone and the alpha-hydroxy carbons of the substrate (C em leader O approximately 3.0 A), which is optimal for the proton transfer involving these centers. The electrophile that polarizes the substrate, His-95, has close contacts to the substrate's O1 and O2 (N em leader O < or = 3.0 and 2.6 A, respectively). The substrate is conformationally relaxed in the Michaelis complex: the phosphate group is out of the plane of the ketone group, and the hydroxy and ketone oxygen atoms are not in the cisoid configuration. The epsilon ammonium group of the electrophilic Lys-12 is within hydrogen-bonding distance of the substrate's ketone oxygen, the bridging oxygen, and a terminal phosphate's oxygen, suggesting a role for this residue in both catalysis and in controlling the flexibility of active-site loop.
The open reading frame TM1643 of Thermotoga maritima belongs to a large family of proteins, with homologues in bacteria, archaea, and eukaryotes. TM1643 is found in an operon with two other genes that encode enzymes involved in the biosynthesis of NAD. In several bacteria, the gene in the position occupied by TM1643 encodes an aspartate oxidase (NadB), which synthesizes iminoaspartate as a substrate for NadA, the next enzyme in the pathway. The amino acid sequence of TM1643 does not share any recognizable homology with aspartate oxidase or with other proteins of known functions or structures. To help define the biological functions of TM1643, we determined its crystal structure at 2.6A resolution and performed a series of screens for enzymatic function. The structure reveals the presence of an N-terminal Rossmann fold domain with a bound NAD(+) cofactor and a C-terminal alpha+beta domain. The structural information suggests that TM1643 may be a dehydrogenase and the active site of the enzyme is located at the interface between the two domains. The enzymatic characterization of TM1643 revealed that it possesses NAD or NADP-dependent dehydrogenase activity toward l-aspartate but no aspartate oxidase activity. The product of the aspartate dehydrogenase activity is also iminoaspartate. Therefore, our studies demonstrate that two different enzymes, an oxidase and a dehydrogenase, may have evolved to catalyze the first step of NAD biosynthesis in prokaryotes. TM1643 establishes a new class of amino acid dehydrogenases.
The Toll/interleukin-1 receptor (TIR) domains are conserved modules in the intracellular regions of the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). The domains are crucial for the signal transduction by these receptors, through homotypic interactions among the receptor and the downstream adapter TIR domains. Previous studies showed that the BB loop in the structure of the TIR domain forms a prominent conserved feature on the surface and is important for receptor signaling. Here we report the crystal structure of the C713S mutant of the TIR domain of human TLR2. An extensively associated dimer is observed in the crystal structure and mutations of several residues in this dimer interface abolished the function of the receptor. Moreover, the structure shows that the BB loop can adopt different conformations, which are required for the formation of this dimer. This asymmetric dimer might represent the TLR2:TLRx heterodimer in the function of this receptor.
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The BEACH domain is highly conserved in a large family of eukaryotic proteins, and is crucial for their functions in vesicle trafficking, membrane dynamics and receptor signaling. However, it does not share any sequence homology with other proteins. Here we report the crystal structure at 2.9 A resolution of the BEACH domain of human neurobeachin. It shows that the BEACH domain has a new and unusual polypeptide backbone fold, as the peptide segments in its core do not assume regular secondary structures. Unexpectedly, the structure also reveals that the BEACH domain is in extensive association with a novel, weakly conserved pleckstrin-homology (PH) domain. Consistent with the structural analysis, biochemical studies show that the PH and BEACH domains have strong interactions, suggesting they may function as a single unit. Functional studies in intact cells demonstrate the requirement of both the PH and the BEACH domains for activity. A prominent groove at the interface between the two domains may be used to recruit their binding partners.
We report on a series of N-pyrazole, N'-aryl ureas and their mode of binding to p38 mitogen activated protein kinase. Importantly, a key binding domain that is distinct from the adenosine 5'-triphoshate (ATP) binding site is exposed when the conserved activation loop, consisting in part of Asp168-Phe169-Gly170, adopts a conformation permitting lipophilic and hydrogen bonding interactions between this class of inhibitors and the protein. We describe the correlation of the structure-activity relationships and crystallographic structures of these inhibitors with p38. In addition, we incorporated another binding pharmacophore that forms a hydrogen bond at the ATP binding site. This modification affords significant improvements in binding, cellular, and in vivo potencies resulting in the selection of 45 (BIRB 796) as a clinical candidate for the treatment of inflammatory diseases.
The regulation of human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) by ATP and fumarate may be crucial for the metabolism of glutamine for energy production in rapidly proliferating tissues and tumors. Here we report the crystal structure at 2.2 A resolution of m-NAD-ME in complex with ATP, Mn2+, tartronate, and fumarate. Our structural, kinetic, and mutagenesis studies reveal unexpectedly that ATP is an active-site inhibitor of the enzyme, despite the presence of an exo binding site. The structure also reveals the allosteric binding site for fumarate in the dimer interface. Mutations in this binding site abolished the activating effects of fumarate. Comparison to the structure in the absence of fumarate indicates a possible molecular mechanism for the allosteric function of this compound.
The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.
Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO(2) in the presence of divalent cations (Mg(2+), Mn(2+)). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD(+) or NADP(+), but not both. Structural studies of the human mitochondrial NAD(+)-dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP(+)-dependent malic enzyme, in a closed form, in a quaternary complex with NADP(+), Mn(2+), and oxalate. This represents the first structural information on an NADP(+)-dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2'-phosphate group of the NADP(+) cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP(+) selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.