Joel M. Harp

Structural Basis for Bile Acid Binding and Activation of the Nuclear Receptor FXR

The nuclear receptor FXR is the sensor of physiological levels of enterohepatic bile acids, the end products of cholesterol catabolism. Here we report crystal structures of the FXR ligand binding domain in complex with coactivator peptide and two different bile acids. An unusual A/B ring juncture, a feature associated with bile acids and no other steroids, provides ligand discrimination and triggers a pi-cation switch that activates FXR. Helix 12, the activation function 2 of the receptor, adopts the agonist conformation and stabilizes coactivator peptide binding. FXR is able to interact simultaneously with two coactivator motifs, providing a mechanism for enhanced binding of coactivators through intermolecular contacts between their LXXLL sequences. These FXR complexes provide direct insights into the design of therapeutic bile acids for treatment of hyperlipidemia and cholestasis.

Asymmetries in the nucleosome core particle at 2.5 Å resolution

The 2.5 A X-ray crystal structure of the nucleosome core particle presented here provides significant additions to the understanding of the nucleosome, the fundamental unit of chromatin structure. Extensions are made to the structure of the N-terminal histone tails and details are provided on hydration and ion binding. The structure is composed of twofold symmetric molecules, native chicken histone octamer cores and the DNA palindrome, which were expected to form a perfectly twofold symmetric nucleosome core particle. In fact, the result is asymmetric owing to the binding of the DNA to the protein surface and to the packing of the particles in the crystal lattice. An analysis is made of the asymmetries by comparisons both within the nucleosome core particle and to the structure of the histone octamer core of the nucleosome.

The active site of the SET domain is constructed on a knot.

The SET domain contains the catalytic center of lysine methyltransferases that target the N-terminal tails of histones and regulate chromatin function. Here we report the structure of the SET7/9 protein in the absence and presence of its cofactor product, S-adenosyl-L-homocysteine (AdoHcy). A knot within the SET domain helps form the methyltransferase active site, where AdoHcy binds and lysine methylation is likely to occur. A structure-guided comparison of sequences within the SET protein family suggests that the knot substructure and active site environment are conserved features of the SET domain.

Molecular Basis for Cyclooxygenase Inhibition by the Non-steroidal Anti-inflammatory Drug Naproxen

Naproxen ((S)-6-methoxy-α-methyl-2-naphthaleneacetic acid) is a powerful non-selective non-steroidal anti-inflammatory drug that is extensively used as a prescription and over-the-counter medication. Naproxen exhibits gastrointestinal toxicity, but its cardiovascular toxicity may be reduced compared with other drugs in its class. Despite the fact that naproxen has been marketed for many years, the molecular basis of its interaction with cyclooxygenase (COX) enzymes is unknown. We performed a detailed study of naproxen-COX-2 interactions using site-directed mutagenesis, structure-activity analysis, and x-ray crystallography. The results indicate that each of the pendant groups of the naphthyl scaffold are essential for COX inhibition, and only minimal substitutions are tolerated. Mutation of Trp-387 to Phe significantly reduced inhibition by naproxen, a result that appears unique to this inhibitor. Substitution of S or CH2 for the O atom of the p-methoxy group yielded analogs that were not affected by the W387F substitution and that exhibited increased COX-2 selectivity relative to naproxen. Crystallization and x-ray analysis yielded structures of COX-2 complexed to naproxen and its methylthio analog at 1.7 and 2.3 Å resolution, respectively. The combination of mutagenesis, structure analysis, and x-ray crystallography provided comprehensive information on the unique interactions responsible for naproxen binding to COX-2.

Crystal Structures of Trypanosoma brucei Sterol 14α-Demethylase and Implications for Selective Treatment of Human Infections

Sterol 14α-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 Å resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.

Structure of the heterodimeric Ecdysone Receptor DNA-binding complex

Ecdysteroids initiate molting and metamorphosis in insects via a heterodimeric receptor consisting of the ecdysone receptor (EcR) and ultraspiracle (USP). The EcR–USP heterodimer preferentially mediates transcription through highly degenerate pseudo‐palindromic response elements, resembling inverted repeats of 5′‐AGGTCA‐3′ separated by 1 bp (IR‐1). The requirement for a heterodimeric arrangement of EcR–USP subunits to bind to a symmetric DNA is unusual within the nuclear receptor superfamily. We describe the 2.24 Å structure of the EcR–USP DNA‐binding domain (DBD) heterodimer bound to an idealized IR‐1 element. EcR and USP use similar surfaces, and rely on the deformed minor groove of the DNA to establish protein–protein contacts. As retinoid X receptor (RXR) is the mammalian homolog of USP, we also solved the 2.60 Å crystal structure of the EcR–RXR DBD heterodimer on IR‐1 and found the dimerization and DNA‐binding interfaces to be the same as in the EcR–USP complex. Sequence alignments indicate that the EcR–RXR heterodimer is an important model for understanding how the FXR–RXR heterodimer binds to IR‐1 sites.

Macromolecular Crystal Annealing: Overcoming Increased Mosaicity Associated with Cryocrystallography

Although cryogenic data collection has become the method of choice for macromolecular crystallography, the flash-cooling step can dramatically increase the mosaicity of some crystals. Macromolecular crystal annealing significantly reduces the mosaicity of flash-cooled crystals without affecting molecular structure. The process, which cycles a flash-cooled crystal to ambient temperature and back to cryogenic temperature, is simple, quick and requires no special equipment. The annealing process has been applied to crystals of several different macromolecules grown from different precipitants and using a variety of cryoprotectants. The protocol for macromolecular crystal annealing also has been applied to restore diffraction from flash-cooled crystals that were mishandled during transfer to or from cryogenic storage. These results will be discussed in relation to crystal mosaicity and effects of radiation damage in flash-cooled crystals.

Macromolecular crystal annealing: evaluation of techniques and variables

Additional examples of successful application of macromolecular crystal annealing are presented. A qualitative evaluation of variables related to the annealing process was conducted using a variety of macromolecular crystals to determine in which cases parameters may be varied and in which cases the original macromolecular crystal annealing protocol is preferred. A hypothesis is presented relating the solvent content of the crystal to the specific protocol necessary for the successful application of annealing.

X-ray diffraction analysis of crystals containing twofold symmetric nucleosome core particles.

Nucleosome core particles containing a DNA palindrome and purified chicken erythrocyte histone octamer have been reconstituted and crystallized. The dyad symmetry of the palindrome extends the dyad symmetry of the histone octamer to result in a twofold symmetric particle. This ensures that the structure determined by X-ray diffraction will yield a true representation of the DNA strand rather than the twofold averaged structure which would result from using a non-palindromic DNA sequence. The crystals provide isotropic diffraction to 3.2 A with observed reflections extending to d spacings of about 2.8 A using a rotating-anode Cu Kalpha X-ray source. Although the DNA palindrome is a factor contributing to the quality of the diffraction data, another significant factor is an improved preparative technique which enriches for correctly phased nucleosome core particles.

Synthesis and Structure-Activity Relationships of 5,6,7-substituted Pyrazolopyrimidines: Discovery of a novel TSPO PET Ligand for Cancer Imaging

Focused library synthesis and structure-activity relationship development of 5,6,7-substituted pyrazolopyrimidines led to the discovery of 2-(5,7-diethyl-2-(4-(2-fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (6b), a novel translocator protein (TSPO) ligand exhibiting a 36-fold enhancement in affinity compared to another pyrazolopyrimidine-based TSPO ligand, 6a (DPA-714). Radiolabeling with fluorine-18 ((18)F) facilitated production of 2-(5,7-diethyl-2-(4-(2-[(18)F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide ((18)F-6b) in high radiochemical yield and specific activity. In vivo studies of (18)F-6b were performed which illuminated this agent as an improved probe for molecular imaging of TSPO-expressing cancers.