John Chung

Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators

Nuclear hormone receptors are ligand-activated transcription factors that regulate the expression of genes that are essential for development, reproduction and homeostasis. The hormone response is mediated through recruitment of p160 receptor coactivators and the general transcriptional coactivator CBP/p300, which function synergistically to activate transcription. These coactivators exhibit intrinsic histone acetyltransferase activity, function in the remodelling of chromatin, and facilitate the recruitment of RNA polymerase II and the basal transcription machinery. The activities of the p160 coactivators are dependent on CBP. Both coactivators are essential for proper cell-cycle control, differentiation and apoptosis, and are implicated in cancer and other diseases. To elucidate the molecular basis of assembling the multiprotein activation complex, we undertook a structural and thermodynamic analysis of the interaction domains of CBP and the activator for thyroid hormone and retinoid receptors. Here we show that although the isolated domains are intrinsically disordered, they combine with high affinity to form a cooperatively folded helical heterodimer. Our study uncovers a unique mechanism, called ‘synergistic folding’, through which p160 coactivators recruit CBP/p300 to allow transmission of the hormonal signal to the transcriptional machinery.

Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA

We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone1 HN, 15N,13 Cα and13 Cβ and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.

Structural and dynamic characterization of an unfolded state of poplar apo-plastocyanin formed under nondenaturing conditions.

Plastocyanin is a predominantly β‐sheet protein containing a type I copper center. The conformational ensemble of a denatured state of apo‐plastocyanin formed in solution under conditions of low salt and neutral pH has been investigated by multidimensional heteronuclear NMR spectroscopy. Chemical shift assignments were obtained by using three‐dimensional triple‐resonance NMR experiments to trace through‐bond heteronuclear connectivities along the backbone and side chains. The 3JHN,Hα coupling constants, 15N‐edited proton–proton nuclear Overhauser effects (NOEs), and 15N relaxation parameters were also measured for the purpose of structural and dynamic characterization. Most of the residues corresponding to β‐strands in the folded protein exhibit small upfield shifts of the 13Cα and 13CO resonances relative to random coil values, suggesting a slight preference for backbone dihedral angles in the β region of (ϕ,ψ) space. This is further supported by the presence of strong sequential dαN(i, i + 1) NOEs throughout the sequence. The few dNN(i, i + 1) proton NOEs that are observed are mostly in regions that form loops in the native plastocyanin structure. No medium or long‐range NOEs were observed. A short sequence, between residues 59 and 63, was found to populate a nonnative helical conformation in the unfolded state, as indicated by the shift of the 13Cα, 13CO, and 1Hα resonances relative to random coil values and by the decreased values of the 3JHN,Hα coupling constants. The 15N relaxation parameters indicate restriction of motions on a nanosecond timescale in this region. Intriguingly, this helical conformation is present in a sequence that is close to but not in the same location as the single short helix in the native folded protein. The results are consistent with earlier NMR studies of peptide fragments of plastocyanin and confirm that the regions of the sequence that form β‐strands in the native protein spontaneously populate the β‐region of (ϕ,ψ) space under folding conditions, even in the absence of stabilizing tertiary interactions. We conclude that the state of apo‐plastocyanin present under nondenaturing conditions is a noncompact unfolded state with some evidence of nativelike and nonnative local structuring that may be initiation sites for folding of the protein.

Synthesis of chiral α-acetylenic cyclic amines from α-amino acids: Applications to differentially constrained oxotremorine analogues as muscarinic agents

Abstract Application of the Corey-Fuchs reaction on Boc-prolinal, Boc-4-hydroxyprolinal, Bocpiperidinal and Boc-serinal derivatives to give chiral 2-pyrrolidinyl-, 2-piperidinyl- and 4-oxazolidinyl-acetylene derivatives provided rapid access to a number of differentially constrained oxotremorine analogues as muscarinic agents.

Conformationally Constrained Amino Acids.1 on the Synthesis of 4-Substituted 8-Membered Cyclic Tryptophan Derivatives

Abstract The thermal lactamization of 4-(carboethoxy)-methyl-tryptophan ethyl ester (5), prepared from 4-(carboethoxy)methyl-indole 3 via Gilchrist′s method, in refluxing xylene followed by saponification provided the 8-membered cyclic tryptophan derivative 1. Selective reduction of lactam 6 via the iminium ion 9 furnished the cyclic tryptophan derivative 2

A "Magic-Angle" Sample-Spinning Nuclear Magnetic Resonance Spectroscopic Study of Interference Effects in the Nuclear Spin Relaxation of Polymers *

Abstract The proton-coupled, carbon-13 “magic-angle” sample-spinning nuclear magnetic resonance spin-lattice relaxation behavior of poly (cis-isoprene) and poly (cis-butadiene) as a function of temperature is reported. Analysis of the relaxation behavior of the methine carbons reveals strong temporal correlations between the 13CH dipolar and 13C anisotropic chemical shielding interactions. Equations are presented which enable the interpretation of these interferences for non-axially symmetric shielding tensors, subject to cylindrically symmetric reorientation. In the context of these expressions, the results indicate that the methine moiety in each of the two polymers executes relatively rapid, isotropic motions, on the time scale of the carbon-13 Larmor frequency. It is also shown that in certain polymeric systems, the sign of the interference term may change with frequency. This intriguing property provides additional insight into the relative reorientational anisotropies of the low- and high-frequency motions, and the specific case of the spin-lattice relaxation and differential line broadening of the methine carbons in poly (cis-isoprene) and poly (trans-isoprene) is considered in detail.

Unusual Pyrimidine Participation: Efficient Stereoselective Synthesis of Potent Dual Orexin Receptor Antagonist MK-6096

An asymmetric synthesis of dual orexin receptor antagonist MK-6096 (1) is described. Key steps for the trans-2,5-disubstituted piperidinyl ether fragment include a biocatalytic transamination, a trans-selective Mukaiyama aldol, and a regioselective pyridyl SNAr process. The pyrimidyl benzoic acid was synthesized via a Negishi coupling and a nitrile hydrolysis. Coupling of the two fragments via a catalytic T3P-mediated amidation completed the synthesis. Unusual behaviors in the hydrolysis of pyrimidyl benzonitrile and the amide coupling of the pyrimidyl benzoic acid are also described.

Synthesis of antifungal glucan synthase inhibitors from enfumafungin.

An efficient, new, and scalable semisynthesis of glucan synthase inhibitors 1 and 2 from the fermentation product enfumafungin 3 is described. The highlights of the synthesis include a high-yielding ether bond-forming reaction between a bulky sulfamidate 17 and alcohol 4 and a remarkably chemoselective, improved palladium(II)-mediated Corey-Yu allylic oxidation at the highly congested C-12 position of the enfumafungin core. Multi-hundred gram quantities of the target drug candidates 1 and 2 were prepared, in 12 linear steps with 25% isolated yield and 13 linear steps with 22% isolated yield, respectively.

1H,15N,13C-triple resonance NMR of very large systems at 900 MHz.

We provide quantitative signal to noise data and feasibility study at 900 MHz for 1H-15N-13C triple resonance backbone assignment pulse sequences obtained from a medium sized 2H, 13C, 15N labeled protein slowed down in glycerol-water solution to mimic relaxation and spectroscopic properties of a much larger protein system with macromolecular tumbling correlation time of 52 and 80 ns, respectively, at 296 and 283 K (corresponding to molecular weights of 130 and 250 kDa). Comparisons of several different schemes for transferring magnetization from proton to nitrogen and back to proton confirms Yang and Kays 1999 prediction that avoiding the unfavorable relaxation properties of 1H-15N multiple quantum coherence in the TROSY phase cycle of the final 15N-1H transfer before acquisition is crucial for maximal sensitivity from these very large molecular weight systems. We also show results which confirm some predictions regarding the superiority of TROSY at 900 MHz vs. 800 MHz especially as the molecular weights become very large.

Iron-57 nuclear magnetic resonance spectroscopic study of alkyl isocyanide myoglobins and a comparison of the 57Fe chemical-shift anisotropies of alkyl isocyanide myoglobins, carbonmonoxymyoglobin, ferrocytochrome c, and [57Fe(bipy)3]X2.5H2O (X=Cl, Br, I)

Abstract The 57Fe nuclear magnetic resonance spectra and spin-lattice relaxation times (T1) of ethyl isocyanide (EtNC), isopropyl isocyanide (iPrNC), and n-butyl isocyanide (n-BuNC) ligated ferrous myoglobins (∼ 12 mM, pH 7.1, 22°C) at 8.45 T (corresponding to a 57Fe Larmor frequency of 11.7 MHz) have been obtained. The isotropic chemical shifts are 9223, 9257, and 9238 ppm downfield from Fe(CO)5, which yields chemical-shift anisotropies, ∥δ∥ − δ∥∥, of 1288, 1260, and 1205 ppm, for the EtNC, and n-BuNC species, respectively. The T1 values (of about 140 ms) are very much longer than those found previously for carbonmonoxymyoglobin (∼ 17 ms) and are consistent with a change in sign of the chemical-shift tensor upon moving from carbonmonoxymyoglobin to ferrocytochrome c, as previously postulated by L. Baltzer, (J. Am. Chem. Soc. 109, 3479 (1987)). The solution- and solid-state 57Fe NMR chemical shifts of [57Fe(bipy)3]X2 · 5H2O (X  Cl, Br, I) complexes are also reported, and for the solids it is found that δ∥ = δ∥ = δi = 11,854, 11,721, and 11,635 ppm, for X  Cl, Br, and I, respectively. T1 values for the solids are in the range ∼ 1–5 s, and are probably dominated by electron exchange with paramagnetic impurities.