Michael D. Feese

The mechanism of topoisomerase I poisoning by a camptothecin analog

We report the x-ray crystal structure of human topoisomerase I covalently joined to double-stranded DNA and bound to the clinically approved anticancer agent Topotecan. Topotecan mimics a DNA base pair and binds at the site of DNA cleavage by intercalating between the upstream (−1) and downstream (+1) base pairs. Intercalation displaces the downstream DNA, thus preventing religation of the cleaved strand. By specifically binding to the enzyme–substrate complex, Topotecan acts as an uncompetitive inhibitor. The structure can explain several of the known structure–activity relationships of the camptothecin family of anticancer drugs and suggests that there are at least two classes of mutations that can produce a drug-resistant enzyme. The first class includes changes to residues that contribute to direct interactions with the drug, whereas a second class would alter interactions with the DNA and thereby destabilize the drug-binding site.

Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment

The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO163) to a 2.5-Å resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO163 has an antiparallel four-helix bundle fold. The neutralizing Fab mainly recognizes the C–D crossover loop containing the species invariant residue Q111. Titration calorimetric experiments show that hTPO163 interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 × 109 M–1 and 1.1 × 106 M–1. The presence of the neutralizing Fab did not inhibit binding of hTPO163 to soluble c-Mpl fragments, but the lower-affinity binding disappeared. Together with prior genetic data, these define the structure–function relationships in TPO and the activation scheme of c-Mpl.

Design of an engineered N-terminal HIV-1 gp41 trimer with enhanced stability and potency

HIV fusion is mediated by a conformational transition in which the C‐terminal region (HR2) of gp41 interacts with the N‐terminal region (HR1) to form a six‐helix bundle. Peptides derived from the HR1 form a well‐characterized, trimeric coiled‐coil bundle in the presence of HR2 peptides, but there is little structural information on the isolated HR1 trimer. Using protein design, we have designed synthetic HR1 peptides that form soluble, thermostable HR1 trimers. In vitro binding of HR2 peptides to the engineered trimer suggests that the design strategy has not significantly impacted the ability to form the six‐helix bundle. The peptides have enhanced antiviral activity compared to wild type, with up to 30‐fold greater potency against certain viral isolates. In vitro passaging was used to generate HR1‐resistant virus and the observed resistance mutations map to the HR2 region of gp41, demonstrating that the peptides block the fusion process by binding to the viral HR2 domain. Interestingly, the activity of the HR2 fusion inhibitor, enfuvirtide (ENF), against these resistant viruses is maintained or improved up to fivefold. The 1.5 Å crystal structure of one of these designs has been determined, and we show that the isolated HR1 is very similar to the conformation of the HR1 in the six‐helix bundle. These results provide an initial model of the pre‐fusogenic state, are attractive starting points for identifying novel fusion inhibitors, and offer new opportunities for developing HIV therapeutics based on HR1 peptides.

AutoDrug: fully automated macromolecular crystallography workflows for fragment-based drug discovery.

AutoDrug is software based upon the scientific workflow paradigm that integrates the Stanford Synchrotron Radiation Lightsource macromolecular crystallography beamlines and third-party processing software to automate the crystallography steps of the fragment-based drug-discovery process. AutoDrug screens a cassette of fragment-soaked crystals, selects crystals for data collection based on screening results and user-specified criteria and determines optimal data-collection strategies. It then collects and processes diffraction data, performs molecular replacement using provided models and detects electron density that is likely to arise from bound fragments. All processes are fully automated, i.e. are performed without user interaction or supervision. Samples can be screened in groups corresponding to particular proteins, crystal forms and/or soaking conditions. A single AutoDrug run is only limited by the capacity of the sample-storage dewar at the beamline: currently 288 samples. AutoDrug was developed in conjunction with RestFlow, a new scientific workflow-automation framework. RestFlow simplifies the design of AutoDrug by managing the flow of data and the organization of results and by orchestrating the execution of computational pipeline steps. It also simplifies the execution and interaction of third-party programs and the beamline-control system. Modeling AutoDrug as a scientific workflow enables multiple variants that meet the requirements of different user groups to be developed and supported. A workflow tailored to mimic the crystallography stages comprising the drug-discovery pipeline of CoCrystal Discovery Inc. has been deployed and successfully demonstrated. This workflow was run once on the same 96 samples that the group had examined manually and the workflow cycled successfully through all of the samples, collected data from the same samples that were selected manually and located the same peaks of unmodeled density in the resulting difference Fourier maps.

Crystallization of the functional domain of human thrombopoietin using an antigen-binding fragment derived from neutralizing monoclonal antibody

Thrombopoietin (TPO) is a cytokine which primarily stimulates megakaryocytopoiesis and thrombopoiesis. The functional domain of TPO (TPO(163)) consisting of the N-terminal 163 amino acids was prepared and crystallized. Since the crystallization of TPO(163) was unsuccessful using the standard screening methods, a Fab fragment derived from a neutralizing monoclonal antibody was used for crystallization. It was found that the TPO(163)-Fab complex crystallized reproducibly in 0.1 M potassium phosphate buffer pH 6.0 containing 20-25% polyethylene glycol 4000. Thin crystals (0.2 x 0.2 x 0.02 mm) grew in two space groups: P2(1), with unit-cell parameters a = 133.20, b = 46.71, c = 191.47 A, beta = 90.24 degrees, and C2, with unit-cell parameters a = 131.71, b = 46.48, c = 184.63 A, beta = 90.42 degrees. The results of a molecular-replacement analysis indicate that the Fab molecules interact with each other and provide a suitable interface for crystallization.

Substrate recognition mechanism of a glycosyltrehalose trehalohydrolase from Sulfolobus solfataricus KM1.

Glycosyltrehalose trehalohydrolase (GTHase) is an α‐amylase that cleaves the α‐1,4 bond adjacent to the α‐1,1 bond of maltooligosyltrehalose to release trehalose. To investigate the catalytic and substrate recognition mechanisms of GTHase, two residues, Asp252 (nucleophile) and Glu283 (general acid/base), located at the catalytic site of GTHase were mutated (Asp252→Ser (D252S), Glu (D252E) and Glu283→Gln (E283Q)), and the activity and structure of the enzyme were investigated. The E283Q, D252E, and D252S mutants showed only 0.04, 0.03, and 0.6% of enzymatic activity against the wild‐type, respectively. The crystal structure of the E283Q mutant GTHase in complex with the substrate, maltotriosyltrehalose (G3‐Tre), was determined to 2.6‐Å resolution. The structure with G3‐Tre indicated that GTHase has at least five substrate binding subsites and that Glu283 is the catalytic acid, and Asp252 is the nucleophile that attacks the C1 carbon in the glycosidic linkage of G3‐Tre. The complex structure also revealed a scheme for substrate recognition by GTHase. Substrate recognition involves two unique interactions: stacking of Tyr325 with the terminal glucose ring of the trehalose moiety and perpendicularly placement of Trp215 to the pyranose rings at the subsites −1 and +1 glucose.

Crystallographic Insight Into the Mechanism of Drug-Induced Topoisomerase I DNA Damage

Topoisomerase I (TOP-I) is an essential eukaryotic enzyme that acts to remove supercoils generated during transcription and DNA replication (1). Because of the size of the eukaryotic chromosome, removal of these supercoils can only be accomplished locally by introducing breaks into the DNA helix. Being a type 1 enzyme, TOP-I mediates DNA relaxation by creating a transient, single-strand break in one strand of the DNA duplex. This transient nicking allows the broken strand to rotate around its intact complement, effectively removing local supercoils. Strand nicking results from the transesterification of an active-site tyrosine (Tyr723 in the human TOP-I) at a DNA phosphodiester bond forming a 3′-phosphotyrosine covalent enzyme-DNA complex. The covalent intermediate is reversed when the released 5′-OH of the broken strand reattacks the phosphotyrosine intermediate in a second transesterification reaction (1). The rate of relegation is normally much faster than is the rate of cleavage (2). This ensures that the steady state concentration of the covalent 3′-phosphotyrosyl TOP-I-DNA complex is extremely low. Several DNA lesions and drugs, however, have been shown to stabilize the covalent 3′-phosphotyrosyl intermediate (3). For example, camptothecin (CPT) is a natural product that was originally discovered because of its antitumor activity (4) and was later demonstrated to promote the accumulation of TOP-I-DNA adducts in vitro and in vivo (5,6). It is generally believed that CPTs act to convert TOP-I into a DNA-damaging agent by binding the covalent 3′-phosphotyrosyl intermediate and, specifically, blocking DNA relegation (7, 8). Topo I is the sole intramolecular target of CPT and the cytotoxic effects of CPT poisoning are S-phase-specific (9). Both in vitro and in vivo data support the idea that during DNA replication, the replication complex can collide with the “trapped” TOP-I-DNA complex, resulting in a double-strand break and subsequent apoptotic cell death (10). Presumably, these compounds have anticancer activity because rapidly dividing cells (e.g., cancer cells) enter S-phase more frequently than do normal cells.