Kwang Yeon Hwang

Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125

The c‐jun N‐terminal kinase (JNK) signaling pathway is regulated by JNK‐interacting protein‐1 (JIP1), which is a scaffolding protein assembling the components of the JNK cascade. Overexpression of JIP1 deactivates the JNK pathway selectively by cytoplasmic retention of JNK and thereby inhibits gene expression mediated by JNK, which occurs in the nucleus. Here, we report the crystal structure of human JNK1 complexed with pepJIP1, the peptide fragment of JIP1, revealing its selectivity for JNK1 over other MAPKs and the allosteric inhibition mechanism. The van der Waals contacts by the three residues (Pro157, Leu160, and Leu162) of pepJIP1 and the hydrogen bonding between Glu329 of JNK1 and Arg156 of pepJIP1 are critical for the selective binding. Binding of the peptide also induces a hinge motion between the N‐ and C‐terminal domains of JNK1 and distorts the ATP‐binding cleft, reducing the affinity of the kinase for ATP. In addition, we also determined the ternary complex structure of pepJIP1‐bound JNK1 complexed with SP600125, an ATP‐competitive inhibitor of JNK, providing the basis for the JNK specificity of the compound.

Structure of the catalytic domain of human phosphodiesterase 5 with bound drug molecules

Phosphodiesterases (PDEs) are a superfamily of enzymes that degrade the intracellular second messengers cyclic AMP and cyclic GMP. As essential regulators of cyclic nucleotide signalling with diverse physiological functions, PDEs are drug targets for the treatment of various diseases, including heart failure, depression, asthma, inflammation and erectile dysfunction. Of the 12 PDE gene families, cGMP-specific PDE5 carries out the principal cGMP-hydrolysing activity in human corpus cavernosum tissue. It is well known as the target of sildenafil citrate (Viagra) and other similar drugs for the treatment of erectile dysfunction. Despite the pressing need to develop selective PDE inhibitors as therapeutic drugs, only the cAMP-specific PDE4 structures are currently available. Here we present the three-dimensional structures of the catalytic domain (residues 537–860) of human PDE5 complexed with the three drug molecules sildenafil, tadalafil (Cialis) and vardenafil (Levitra). These structures will provide opportunities to design potent and selective PDE inhibitors with improved pharmacological profiles.

High-resolution crystal structure of the non-specific lipid-transfer protein from maize seedlings

BACKGROUND The movement of lipids between membranes is aided by lipid-transfer proteins (LTPs). Some LTPs exhibit broad specificity, transferring many classes of lipids, and are termed non-specific LTPs (ns-LTPs). Despite their apparently similar mode of action, no sequence homology exists between mammalian and plant ns-LTPs and no three-dimensional structure has been reported for any plant ns-LTP. RESULTS We have determined the crystal structure of ns-LTP from maize seedlings by multiple isomorphous replacement and refined the structure to 1.9 A resolution. The protein comprises a single compact domain with four alpha-helices and a long C-terminal region. The eight conserved cysteines form four disulfide bridges (assigned as Cys4-Cys52, Cys14-Cys29, Cys30-Cys75, and Cys50-Cys89) resolving the ambiguity that remained from the chemical determination of pairings in the homologous protein from castor bean. Two of the bonds, Cys4-Cys52 and Cys50-Cys89, differ from what would have been predicted from sequence alignment with soybean hydrophobic protein. The complex between maize ns-LTP and hexadecanoate (palmitate) has also been crystallized and its structure refined to 1.8 A resolution. CONCLUSIONS The fold of maize ns-LTP places it in a new category of all-alpha-type structure, first described for soybean hydrophobic protein. In the absence of a bound ligand, the protein has a tunnel-like hydrophobic cavity, which is large enough to accommodate a long fatty acyl chain. In the structure of the complex with palmitate, most of the acyl chain is buried inside this hydrophobic cavity.

The crystal structure of flap endonuclease-1 from Methanococcus jannaschii.

Flap endonuclease-1 (FEN-1), a structure specific nuclease, is an essential enzyme for eukaryotic DNA replication and repair. The crystal structure of FEN-1 from Methanococcus jannaschii, determined at 2.0 Å resolution, reveals an active site with two metal ions residing on top of a deep cleft where several conserved acidic residues are clustered. Near the active site, a long flexible loop comprised of many basic and aromatic residues forms a hole large enough to accommodate the DNA substrate. Deletion mutations in this loop significantly decreased the nuclease activity and specificity of FEN-1, suggesting that the loop is critical for recognition and cleavage of the junction between single and double-stranded regions of flap DNA.

Structural basis for cold adaptation. Sequence, biochemical properties, and crystal structure of malate dehydrogenase from a psychrophile Aquaspirillium arcticum.

Aquaspillium arcticum is a psychrophilic bacterium that was isolated from arctic sediment and grows optimally at 4 °C. We have cloned, purified, and characterized malate dehydrogenase from A. arcticum (Aa MDH). We also have determined the crystal structures of apo-Aa MDH, Aa MDH·NADH binary complex, and Aa MDH·NAD·oxaloacetate ternary complex at 1.9-, 2.1-, and 2.5-Å resolutions, respectively. The Aa MDH sequence is most closely related to the sequence of a thermophilic MDH fromThermus flavus (Tf MDH), showing 61% sequence identity and over 90% sequence similarity. Stability studies show that Aa MDH has a half-life of 10 min at 55 °C, whereas Tf MDH is fully active at 90 °C for 1 h. Aa MDH shows 2–3-fold higher catalytic efficiency compared with a mesophilic or a thermophilic MDH at the temperature range 4–10 °C. Structural comparison of Aa MDH and Tf MDH suggests that the increased relative flexibility of active site residues, favorable surface charge distribution for substrate and cofactor, and the reduced intersubunit ion pair interactions may be the major factors for the efficient catalytic activity of Aa MDH at low temperatures.

Structure-based identification of a novel NTPase from Methanococcus jannaschii.

Almost half of the entire set of predicted genomic products from Methanococcus jannaschii are classified as functionally unknown hypothetical proteins. We present a structure-based identification of the biochemical function of a protein with an as yet unknown function from a M. jannaschii gene, Mj0226. The crystal structure of Mj0226 protein determined at 2.2 Å resolution reveals that the protein is a homodimer and each monomer folds into an elongated α/β structure of a new fold family. Comparisons of Mj0226 protein with protein structures in the database, however, indicate that one part of the protein is homologous to some of the nucleotide-binding proteins. Biochemical analysis shows that Mj0226 protein is a novel nucleotide triphosphatase that can efficiently hydrolyze nonstandard nucleotides such as XTP to XMP or ITP to IMP, but not the standard nucleotides, in the presence of Mg2+ or Mn2+ ions.

Crystal structure of carboxylesterase from Pseudomonas fluorescens, an α/β hydrolase with broad substrate specificity

Background: A group of esterases, classified as carboxylesterases, hydrolyze carboxylic ester bonds with relatively broad substrate specificity and are useful for stereospecific synthesis and hydrolysis of esters. One such carboxylesterase from Pseudomonas fluorescens is a homodimeric enzyme, consisting of 218-residue subunits. It shows a limited sequence similarity to some members of the α/β hydrolase superfamily. Although crystal structures of a number of serine esterases and lipases have been reported, structural information on carboxylesterases is very limited. This study was undertaken in order to provide such information and to understand a structural basis for the substrate specificity of this carboxylesterase. Results: In this study, the crystal structure of carboxylesterase from P. fluorescens has been determined by the isomorphous replacement method and refined to 1.8 A resolution. Each subunit consists of a central seven-stranded β sheet flanked by six α helices. The structure reveals the catalytic triad as Ser114‐His199‐Asp168. The structure of the enzyme in complex with the inhibitor phenylmethylsulfonyl fluoride has also been determined and refined to 2.5 A. The inhibitor is covalently attached to Ser114 of both subunits, with the aromatic ring occupying a hydrophobic site defined by the aliphatic sidechains of Leu23, Ile58, Ile70, Met73 and Val170. No large structural changes are observed between the free and inhibitorbound structures. Conclusions: Carboxylesterase from P. fluorescens has the α/β hydrolase fold and the Ser‐His‐Asp catalytic triad. The active-site cleft in each subunit is formed by the six loops covering the catalytic serine residue. Three of the active-site loops in each subunit are involved in a head-to-head subunit interaction to form a dimer; it may be these extra structural elements, not seen in other esterases, that account for the inability of carboxylesterase to hydrolyze long chain fatty acids. As a result of dimerization, the active-site clefts from the two subunits merge to form holes in the dimer. The active-site clefts are relatively open and thus the catalytic residues are exposed to the solvent. An oxyanion hole, formed by nitrogen atoms of Leu23 and Gln115, is present in both the free and inhibitor-bound structures. An open active site, as well as a large binding pocket for the acid part of substrates, in P. fluorescens carboxylesterase may contribute to its relatively broad substrate specificity.

Structure and mechanism of glutamate racemase from Aquifex pyrophilus.

Glutamate racemase (MurI) is responsible for the synthesis of D-glutamate, an essential building block of the peptidoglycan layer in bacterial cell walls. The crystal structure of glutamate racemase from Aquifex pyrophilus, determined at 2.3 Å resolution, reveals that the enzyme forms a dimer and each monomer consists of two α/β fold domains, a unique structure that has not been observed in other racemases or members of an enolase superfamily. A substrate analog, D-glutamine, binds to the deep pocket formed by conserved residues from two monomers. The structural and mutational analyses allow us to propose a mechanism of metal cofactor-independent glutamate racemase in which two cysteine residues are involved in catalysis.

Ursolic acid is a PPAR-α agonist that regulates hepatic lipid metabolism

In this study, we confirmed that ursolic acid, a plant triterpenoid, activates peroxisome proliferator-activated receptor (PPAR)-α in vitro. Surface plasmon resonance and time-resolved fluorescence resonance energy transfer analyses do not show direct binding of ursolic acid to the ligand-binding domain of PPAR-α; however, ursolic acid enhances the binding of PPAR-α to the peroxisome proliferator response element in PPAR-α-responsive genes, alters the expression of key genes in lipid metabolism, significantly reducing intracellular triglyceride and cholesterol concentrations in hepatocytes. Thus, ursolic acid is a PPAR-α agonist that regulates the expression of lipid metabolism genes, but it is not a direct ligand of PPAR-α.

Structural studies of human brain-type creatine kinase complexed with the ADP-Mg2+-NO3- -creatine transition-state analogue complex

Creatine kinase is a member of the phosphagen kinase family, which catalyzes the reversible phosphoryl transfer reaction that occurs between ATP and creatine to produce ADP and phosphocreatine. Here, three structural aspects of human‐brain‐type‐creatine‐kinase (hBB‐CK) were identified by X‐ray crystallography: the ligand‐free‐form at 2.2 Å; the ADP–Mg2+, nitrate, and creatine complex (transition‐state‐analogue complex; TSAC); and the ADP–Mg2+‐complex at 2.0 Å. The structures of ligand‐bound hBB‐CK revealed two different monomeric states in a single homodimer. One monomer is a closed form, either bound to TSAC or the ADP–Mg2+‐complex, and the second monomer is an unliganded open form. These structural studies provide a detailed mechanism indicating that the binding of ADP–Mg2+ alone may trigger conformational changes in hBB‐CK that were not observed with muscle‐type‐CK.