Barry C. Finzel

High-resolution crystal structure of cytochrome P450cam.

The crystal structure of Pseudomonas putida cytochrome P450cam with its substrate, camphor, bound has been refined to R = 0.19 at a normal resolution of 1.63 A. While the 1.63 A model confirms our initial analysis based on the 2.6 A model, the higher resolution structure has revealed important new details. These include a more precise assignment of sequence to secondary structure, the identification of three cis-proline residues, and a more detailed picture of substrate-protein interactions. In addition, 204 ordered solvent molecules have been found, one of which appears to be a cation. The cation stabilizes an unfavorable polypeptide conformation involved in forming part of the active site pocket, suggesting that the cation may be the metal ion binding site associated with the well-known ability of metal ions to enhance formation of the enzyme-substrate complex. Another unusual polypeptide conformation forms the proposed oxygen-binding pocket. A localized distortion and widening of the distal helix provides a pocket for molecular oxygen. An intricate system of side-chain to backbone hydrogen bonds aids in stabilizing the required local disruption in helical geometry. Sequence homologies strongly suggest a common oxygen-binding pocket in all P450 species. Further sequence comparisons between P450 species indicate common three-dimensional structures with changes focused in a region of the molecule postulated to be associated with the control of substrate specificity.

Crystal structure of recombinant human interleukin-1β at 2·0 Å resolution

Abstract The crystal structure of recombinant human interleukin-1β (IL-1β) has been determined at 2·0 A resolution and refined to a crystallographic R-factor of 0·19. Three heavy-atom derivatives were identified and used for multiple isomorphous replacement phasing. Interpretation of the resulting electron density map revealed a structure in which there are 12 antiparallel β-strands and no α-helix. The single 153-residue polypeptide chain is folded into a six-stranded β-barrel similar in architecture to the Kunitz-type trypsin inhibitor found in soybeans. The molecule displays approximate 3-fold symmetry about the axis of the β-barrel. Each successive pair of component strands of the barrel brackets an extensive sequence outside the barrel that includes an additional pair of β-strands and a prominent loop. Together, these three external segments conceal much of the perimeter and one end of the barrel, leaving only the end supporting the chain termini fully exposed. The structure can be used to identify portions of the polypeptide chain that are exposed on the surface of the molecule, some of which must be epitopes recognized by interleukin-1β receptors.

Recent results and new hardware developments for protein crystal growth in microgravity

Abstract Protein crystal growth experiments have been performed on 16 space shuttle missions since April 1985. The initial experiments used vapor diffusion crystallization techniques similar to those used in laboratories for earth-based experiments. More recent experiments have assessed temperature-induced crystallization as an alternative method for growing high quality protein crystals in microgravity. Results from both vapor-diffusion and temperature-induced crystallization experiments indicate that protein crystals grown in microgravity may be larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth.

Inhibition of Mycobacterium tuberculosis Transaminase BioA by Aryl Hydrazines and Hydrazides.

7,8‐Diaminopelargonic acid synthase (BioA) of Mycobacterium tuberculosis is a recently validated target for therapeutic intervention in the treatment of tuberculosis (TB). Using biophysical fragment screening and structural characterization of compounds, we have identified a potent aryl hydrazine inhibitor of BioA that reversibly modifies the pyridoxal‐5′‐phosphate (PLP) cofactor, forming a stable quinonoid. Analogous hydrazides also form covalent adducts that can be observed crystallographically but are incapable of inactivating the enzyme. In the X‐ray crystal structures, small molecules induce unexpected conformational remodeling in the substrate binding site. We compared these conformational changes to those induced upon binding of the substrate (7‐keto‐8‐aminopelargonic acid), and characterized the inhibition kinetics and the X‐ray crystal structures of BioA with the hydrazine compound and analogues to unveil the mechanism of this reversible covalent modification.

Target-based identification of whole-cell active inhibitors of biotin biosynthesis in Mycobacterium tuberculosis.

Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counterscreen in biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counterscreen proved crucial to filter out compounds whose whole-cell activity was off target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were cocrystallized with BioA to provide a framework for future structure-based drug design efforts.

Kinetic and structural characterization of caspase-3 and caspase-8 inhibition by a novel class of irreversible inhibitors.

Because of their central role in programmed cell death, the caspases are attractive targets for developing new therapeutics against cancer and autoimmunity, myocardial infarction and ischemic damage, and neurodegenerative diseases. We chose to target caspase-3, an executioner caspase, and caspase-8, an initiator caspase, based on the vast amount of information linking their functions to diseases. Through a structure-based drug design approach, a number of novel beta-strand peptidomimetic compounds were synthesized. Kinetic studies of caspase-3 and caspase-8 inhibition were carried out with these urazole ring-containing irreversible peptidomimetics and a known irreversible caspase inhibitor, Z-VAD-fmk. Using a stopped-flow fluorescence assay, we were able to determine individual kinetic parameters of caspase-3 and caspase-8 inhibition by these inhibitors. Z-VAD-fmk and the peptidomimetic inhibitors inhibit caspase-3 and caspase-8 via a three-step kinetic mechanism. Inhibition of both caspase-3 and caspase-8 by Z-VAD-fmk and of caspase-3 by the peptidomimetic inhibitors proceeds via two rapid equilibrium steps followed by a relatively fast inactivation step. However, caspase-8 inhibition by the peptidomimetics goes through a rapid equilibrium step, a slow-binding reversible step, and an extremely slow inactivation step. The crystal structures of inhibitor complexes of caspases-3 and -8 validate the design of the inhibitors by illustrating in detail how they mimic peptide substrates. One of the caspase-8 structures also shows binding at a secondary, allosteric site, providing a possible route to the development of noncovalent small molecule modulators of caspase activity.

Fragment-Based Identification of an Inducible Binding Site on Cell Surface Receptor CD44 for the Design of Protein-Carbohydrate Interaction Inhibitors.

Selective inhibitors of hyaluronan (HA) binding to the cell surface receptor CD44 will have value as probes of CD44-mediated signaling and have potential as therapeutic agents in chronic inflammation, cardiovascular disease, and cancer. Using biophysical binding assays, fragment screening, and crystallographic characterization of complexes with the CD44 HA binding domain, we have discovered an inducible pocket adjacent to the HA binding groove into which small molecules may bind. Iterations of fragment combination and structure-driven design have allowed identification of a series of 1,2,3,4-tetrahydroisoquinolines as the first nonglycosidic inhibitors of the CD44-HA interaction. The affinity of these molecules for the CD44 HA binding domain parallels their ability to interfere with CD44 binding to polymeric HA in vitro. X-ray crystallographic complexes of lead compounds are described and compared to a new complex with a short HA tetrasaccharide, to establish the tetrahydroisoquinoline pharmacophore as an attractive starting point for lead optimization.

LORE : EXPLOITING DATABASE OF KNOWN STRUCTURES

The collection of known protein structures contains a wealth of information, not only about small building blocks of proteins, but also about the way these elements assemble in real structures. Both types of information can be valuable to the modeler or crystallographer. With the software known as LORE, we provide substructure search and coordinate manipulation tools that place a wide variety of different types of structural information at the users fingertips. Substructures, varying in complexity from small segments to complicated elements of a protein fold, can be identified and used in a wide range of applications, from density interpretation to protein engineering. To our knowledge, no other database modeling software provides such generalized coordinate manipulation capabilities. We have found the software to be a valuable addition to our molecular modeling tools.

Conserved core substructures in the overlay of protein-ligand complexes

The method of conserved core substructure matching (CSM) for the overlay of protein-ligand complexes is described. The method relies upon distance geometry to align structurally similar substructures without regard to sequence similarity onto substructures from a reference protein empirically selected to include key determinants of binding site location and geometry. The error in ligand position is reduced in reoriented ensembles generated with CSM when compared to other overlay methods. Since CSM can only succeed when the selected core substructure is geometrically conserved, misalignments only rarely occur. The method may be applied to reliably overlay large numbers of protein-ligand complexes in a way that optimizes ligand position at a specific binding site or subsite or to align structures from large and diverse protein families where the conserved binding site is localized to only a small portion of either protein. Core substructures may be complex and must be chosen with care. We have created a database of empirically selected core substructures to demonstrate the utility of CSM alignment of ligand binding sites in important drug targets. A Web-based interface can be used to apply CSM to align large collections of protein-ligand complexes for use in drug design using these substructures or to evaluate the use of alternative core substructures that may then be shared with the larger user community. Examples show the benefit of CSM in the practice of structure-based drug design.

Structural characterization of human histidine triad nucleotide-binding protein 2, a member of the histidine triad superfamily.

The histidine triad proteins (HITs) constitute a large and ubiquitous superfamily of nucleotide hydrolases. The human histidine triad nucleotide‐binding proteins (hHints) are a distinct class of HITs noted for their acyl‐AMP hydrolase and phosphoramidase activity. The first high‐resolution crystal structures of hHint2 with and without bound AMP are described. The differences between hHint2 and previously known HIT family protein structures are discussed. HIT family enzymes have historically been divided into five classes based on their catalytic specificity: Hint, fragile HIT protein, galactose‐1‐phosphate uridylyltransferase, DcpS and aprataxin. However, although several structures exist for the enzymes in these classes, the endogenous substrates of many of these enzymes have not been identified or biochemically characterized. To better understand the structural relationships of the HIT enzymes, a structure‐based phylogeny was constructed that resulted in the identification of several new putative HIT clades with potential acyl‐AMP hydrolase and phosphoramidase activity.