Sung-Hou Kim

Sparse matrix sampling: a screening method for crystallization of proteins

A set of screening conditions for initial experiments in protein crystallization has been developed, tested, and is herein presented. These solution and precipitant conditions are empirically derived based on known or published crystallization conditions of various proteins in the past, so as to sample as large a range of buffer, pH, additive and precipitant variables as possible, using small amounts of proteins. The 50 crystallization conditions have been tested on 15 previously crystallized proteins, all of which were also crystallized in at least one form by this screen. This method is also shown to be highly successful in the crystallization of proteins which had not previously been crystallized.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.

BRAFV600E is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting “active” protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-RafV600E with an IC50 of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-RafV600E kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-RafV600E-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-RafV600E-positive cells. In B-RafV600E-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-RafV600E-driven tumors.

Electron Transfer by Domain Movement in Cytochrome Bc1

The cytochrome bc1 is one of the three major respiratory enzyme complexes residing in the inner mitochondrial membrane. Cytochrome bc1 transfers electrons from ubiquinol to cytochrome c and uses the energy thus released to form an electrochemical gradient across the inner membrane. Our X-ray crystal structures of the complex from chicken, cow and rabbit in both the presence and absence of inhibitors of quinone oxidation, reveal two different locations for the extrinsic domain of one component of the enzyme, an iron–sulphur protein. One location is close enough to the supposed quinol oxidation site to allow reduction of the Fe–S protein by ubiquinol. The other site is close enough to cytochrome c1 to allow oxidation of the Fe–S protein by the cytochrome. As neither location will allow both reactions to proceed at a suitable rate, the reaction mechanism must involve movement of the extrinsic domain of the Fe–S component in order to shuttle electrons from ubiquinol to cytochrome c1. Such a mechanism has not previously been observed in redox protein complexes.

Crystal structure of a small heat-shock protein

The principal heat-shock proteins that have chaperone activity (that is, they protect newly made proteins from misfolding) belong to five conserved classes: HSP100, HSP90, HSP70, HSP60 and the small heat-shock proteins (sHSPs). The sHSPs can form large multimeric structures and have a wide range of cellular functions, including endowing cells with thermotolerance in vivo, and being able to act as molecular chaperones in vitro; sHSPs do this by forming stable complexes with folding intermediates of their protein substrates,. However, there is little information available about these structures or the mechanism by which substrates are protected from thermal denaturation by sHSPs. Here we report the crystal structure of a small heat-shock protein from Methanococcus jannaschii, a hyperthermophilic archaeon. The monomeric folding unit is a composite β-sandwich in which one of the β-strands comes from a neighbouring molecule. Twenty-four monomers form a hollow spherical complex of octahedral symmetry, with eight trigonal and six square ‘windows’. The sphere has an outer diameter of 120 Å and an inner diameter of 65 Å.

Three-dimensional tertiary structure of yeast phenylalanine transfer RNA

The 3-angstrom electron density map of crystalline yeast phenylalanine transfer RNA has provided us with a complete three-dimensional model which defines the positions of all of the nucleotide residues in the moleclule. The overall features of the molecule are virtually the same as those seen at a resolution of 4 angstroms except that many additional details of tertiary structure are now visualized. Ten types of hydrogen bonding are identified which define the specificity of tertiary interactions. The molecule is also stabilized by considerable stacking of the planar purines and pyrimidines. This tertiary structure explains, in a simple and direct fashion, chemical modification studies of transfer RNA. Since most of the tertiary interactions involve nucleotides which are common to all transfer RNA s, it is likely that this three-dimensional structure provides a basic pattern of folding which may help to clarify the three-dimensional structure of all transfer RNAs.

Crystal structure of the ATP-binding subunit of an ABC transporter.

ABC transporters (also known as traffic ATPases) form a large family of proteins responsible for the translocation of a variety ofcompounds across membranes of both prokaryotes and eukaryotes. The recently completed Escherichia coli genome sequence revealed that the largest family of paralogous E. coli proteins is composed of ABC transporters. Many eukaryotic proteins of medical significance belong to this family, such as the cystic fibrosis transmembrane conductance regulator (CFTR), the P-glycoprotein (or multidrug-resistance protein) and the heterodimeric transporter associated with antigen processing (Tap1–Tap2). Here we report the crystal structure at 1.5 Å resolution of HisP, the ATP-binding subunit of the histidine permease, which is an ABC transporter from Salmonella typhimurium. We correlate the details of this structure with the biochemical, genetic and biophysical properties of the wild-type and several mutant HisP proteins. The structure provides a basis for understanding properties of ABC transporters and of defective CFTR proteins.

Four-helical-bundle structure of the cytoplasmic domain of a serine chemotaxis receptor.

The bacterial chemotaxis receptors are transmembrane receptors with a simple signalling pathway which has elements relevant to the general understanding of signal recognition and transduction across membranes, how signals are relayed between molecules in a pathway, and how adaptation to a persistent signal is achieved. In contrast to many mammalian receptors which signal by oligomerizing upon ligand binding, the chemotaxis receptors are dimeric even in the absence of their ligands, and their signalling does not depend on a monomer–dimer equilibrium. Bacterial chemotaxis receptors are composed of a ligand-binding domain, a transmembrane domain consisting of two helices TM1 and TM2, and a cytoplasmic domain. All known bacterial chemotaxis receptors have a highly conserved cytoplasmic domain, which unites signals from different ligand domains into a single signalling pathway to flagella motors. Here we report the crystal structure of the cytoplasmic domain of a serine chemotaxis receptor of Escherichia coli, which reveals a 200 å-long coiled-coil of two antiparallel helices connected by a ‘U-turn’. Two of these domains form a long, supercoiled, four-helical bundle in the cytoplasmic portion of the receptor.

Inhibition of cyclin-dependent kinases, GSK-3β and CK1 by hymenialdisine, a marine sponge constituent

BACKGROUND Over 2000 protein kinases regulate cellular functions. Screening for inhibitors of some of these kinases has already yielded some potent and selective compounds with promising potential for the treatment of human diseases. RESULTS The marine sponge constituent hymenialdisine is a potent inhibitor of cyclin-dependent kinases, glycogen synthase kinase-3beta and casein kinase 1. Hymenialdisine competes with ATP for binding to these kinases. A CDK2-hymenialdisine complex crystal structure shows that three hydrogen bonds link hymenialdisine to the Glu81 and Leu83 residues of CDK2, as observed with other inhibitors. Hymenialdisine inhibits CDK5/p35 in vivo as demonstrated by the lack of phosphorylation/down-regulation of Pak1 kinase in E18 rat cortical neurons, and also inhibits GSK-3 in vivo as shown by the inhibition of MAP-1B phosphorylation. Hymenialdisine also blocks the in vivo phosphorylation of the microtubule-binding protein tau at sites that are hyperphosphorylated by GSK-3 and CDK5/p35 in Alzheimers disease (cross-reacting with Alzheimers-specific AT100 antibodies). CONCLUSIONS The natural product hymenialdisine is a new kinase inhibitor with promising potential applications for treating neurodegenerative disorders.

Crystal structure of yeast phenylalanine transfer RNA. I. Crystallographic refinement.

We present the results of the Jimal stage of the X-ray crystallographic studies of yeast phenylalanine transfer RNA in an orthorhombic crystal form. The crystal structure of the transfer KNA has been refined by a least-squares procedure to minimize the difference between the observed (F,) and calculated (F’,) structure factors from X-ray diffraction patterns. The final crystallographic discrepancy index, R = IIF,, - Fcl/IF,, is 0.198, based upori 8426 structure factors with magnitudes over twice the estimated standard deviation, corresponding to 96.4% of the complete set of data with resolutions up t,o 2.7 A. During the refinement, bond lengths and angles within each phosphate group and each rmcleoside (base plus sugar) were constrained exactly to their appropriate standard values, while those for the linkages between the nucleosides and phosphates were elastically restrained close to their standard values. The details of the application of the constraint-restraint least-squares (CORELS) refinement method to the crystal structure of yeast phenylalanine tRNA are described in this paper. A complete list of atomic co-ordinates and the rigid group thermal factors are presented. The st,ereochemical details of this structrire and their frictional implications are described in the following paper.

Three-Dimensional Structure of Yeast Phenylalanine Transfer RNA: Folding of the Polynucleotide Chain

At 4 � resolution the polynucleotides in yeast phenylalanine transfer RNA are seen in a series of electron dense masses about 5.8 � apart. These peaks are probably associated with the phosphate groups, while lower levels of electron density between segments of adjacent polynucleotide chains are interpreted as arising from hydrogen-bonded purine-pyrimidine base pairs. It is possible to trace the entire polynucleotide chain with only two minor regions of ambiguity. The polynucleotide chain has a secondary structure consistent with the cloverleaf conformation; however, its folding is different from that proposed in any model. The molecule is made of two double-stranded helical regions oriented at right angles to each other in the shape of an L. One end of the L has the CCA acceptor; the anticodon loop is at the other end, and the dihydrouridine and TψC loops form the corner.