Craig D. Smith

Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase

Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.

Crystal structure of peroxynitrite-modified bovine Cu,Zn superoxide dismutase.

The crystal structure of bovine Cu,Zn superoxide dismutase modified with peroxynitrite (ONOO-) was determined by X-ray diffraction, utilizing the existing three-dimensional model of the native structure deposited in the Brookhaven Protein Data Bank (J. A. Tainer et al., J. Mol. Biol. 160, 181-217, 1982). The native structure and the modified derivative were refined to R factors of 19.0 and 18.7% respectively using diffraction data from 6.0 to 2.5 A. The major result after reaction with peroxynitrite was the appearance of electron density 1.45 A from a single epsilon carbon of Tyr-108, the only tyrosine residue in the sequence. Tyr-108 is a solvent-exposed residue 18 A from the copper atom in the active site. The electron density was consistent with nitration of Tyr-108 at one of the epsilon carbons to form 3-nitrotyrosine. We propose that the nitration occurs in solution by transfer of a nitronium-like species from the active site on one superoxide dismutase dimer to the Tyr-108 of a second dimer.

Mapping the ρ1 GABAC Receptor Agonist Binding Pocket CONSTRUCTING A COMPLETE MODEL

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The GABA receptor type C (GABAC) is a ligand-gated ion channel with pharmacological properties distinct from the GABAA receptor. To date, only three binding domains in the recombinant ρ1 GABAC receptor have been recognized among six potential regions. In this report, using the substituted cysteine accessibility method, we scanned three potential regions previously unexplored in the ρ1 GABAC receptor, corresponding to the binding loops A, E, and F in the structural model for ligand-gated ion channels. The cysteine accessibility scanning and agonist/antagonist protection tests have resulted in the identification of residues in loops A and E, but not F, involved in forming the GABAC receptor agonist binding pocket. Three of these newly identified residues are in a novel region corresponding to the extended stretch of loop E. In addition, the cysteine accessibility pattern suggests that part of loop A and part of loop E have a β-strand structure, whereas loop F is a random coil. Finally, when all of the identified ligand binding residues are mapped onto a three-dimensional homology model of the amino-terminal domain of the ρ1 GABAC receptor, they are facing toward the putative binding pocket. Combined with previous findings, a complete model of the GABAC receptor binding pocket was proposed and discussed in comparison with the GABAA receptor binding pocket.

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.

Protein crystal growth results for shuttle flights STS-26 and STS-29

Abstract Recent advances in protein crystallography have significantly shortened the time and labor required to determine the three-dimensional structures of macromolecules once good crystals are available. Crystal growth has become a major bottleneck in further development of protein crystallography. Proteins and other biological macromolecules are notoriously difficult to crystallize. Even when usable crystals are obtained, the crystals of essentially all proteins and other biological macromolecules are poorly ordered, and diffract to resolutions considerably lower than that available for most crystals of simple organic and inorganic compounds. One promising area of research which is receiving widespread attention is protein crystal growth in the microgravity environment of space. A series of protein crystal growth experiments were performed on US shuttle flight STS-26 in September 1988 and STS-29 in March 1989. These proteins had been studied extensively in crystal growth experiments on earth prior to the microgravity experiments. For those proteins which produced crystals of adequate size, three-dimensional intensity data sets with electronic area detector systems were collected. Comparisons of the microgravity-grown crystals with the best earth-grown crystals obtained in numerous experiments demostrate that the microgravity-grown crystals of these proteins are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions. Analyses of the three-dimensional data sets by relative-Wilson plots indicate that the space-grown crystals are more highly ordered at the molecular level than their earth-grown counterparts.

Defining the Communication between Agonist and Coactivator Binding in the Retinoid X Receptor α Ligand Binding Domain

Background: Some retinoid X receptor (RXR) agonists have potential as cancer drugs. Results: Structures of RXR in complex with two different agonists show similar folds. Dynamics analysis reveals unique ligand-induced dynamics in helices 3, 11, and 12. Conclusion: Two networks of interactions that connect RXR agonists to coactivator binding are defined. Significance: Recognition of common conformational changes and distinguishing dynamics of RXR-selective agonists is necessary for advances in drug design. Retinoid X receptors (RXRs) are obligate partners for several other nuclear receptors, and they play a key role in several signaling processes. Despite being a promiscuous heterodimer partner, this nuclear receptor is a target of therapeutic intervention through activation using selective RXR agonists (rexinoids). Agonist binding to RXR initiates a large conformational change in the receptor that allows for coactivator recruitment to its surface and enhanced transcription. Here we reveal the structural and dynamical changes produced when a coactivator peptide binds to the human RXRα ligand binding domain containing two clinically relevant rexinoids, Targretin and 9-cis-UAB30. Our results show that the structural changes are very similar for each rexinoid and similar to those for the pan-agonist 9-cis-retinoic acid. The four structural changes involve key residues on helix 3, helix 4, and helix 11 that move from a solvent-exposed environment to one that interacts extensively with helix 12. Hydrogen-deuterium exchange mass spectrometry reveals that the dynamics of helices 3, 11, and 12 are significantly decreased when the two rexinoids are bound to the receptor. When the pan-agonist 9-cis-retinoic acid is bound to the receptor, only the dynamics of helices 3 and 11 are reduced. The four structural changes are conserved in all x-ray structures of the RXR ligand-binding domain in the presence of agonist and coactivator peptide. They serve as hallmarks for how RXR changes conformation and dynamics in the presence of agonist and coactivator to initiate signaling.

Crystal structure of human L-isoaspartyl-O-methyl-transferase with S-adenosyl homocysteine at 1.6-Å resolution and modeling of an isoaspartyl-containing peptide at the active site

Spontaneous formation of isoaspartyl residues (isoAsp) disrupts the structure and function of many normal proteins. Protein isoaspartyl methyltransferase (PIMT) reverts many isoAsp residues to aspartate as a protein repair process. We have determined the crystal structure of human protein isoaspartyl methyltransferase (HPIMT) complexed with adenosyl homocysteine (AdoHcy) to 1.6‐Å resolution. The core structure has a nucleotide binding domain motif, which is structurally homologous with the N‐terminal domain of the bacterial Thermotoga maritima PIMT. Highly conserved residues in PIMTs among different phyla are placed at positions critical to AdoHcy binding and orienting the isoAsp residue substrate for methylation. The AdoHcy is completely enclosed within the HPIMT and a conformational change must occur to allow exchange with adenosyl methionine (AdoMet). An ordered sequential enzyme mechanism is supported because C‐terminal residues involved with AdoHcy binding also form the isoAsp peptide binding site, and a change of conformation to allow AdoHcy to escape would preclude peptide binding. Modeling experiments indicated isoAsp groups observed in some known protein crystal structures could bind to the HPIMT active site.

Structure of nicotinic acid mononucleotide adenylyltransferase from Bacillus anthracis

Nicotinic acid mononucleotide adenylyltransferase (NaMNAT; EC 2.7.7.18) is the penultimate enzyme in the biosynthesis of NAD(+) and catalyzes the adenylation of nicotinic acid mononucleotide (NaMN) by ATP to form nicotinic acid adenine dinucleotide (NaAD). This enzyme is regarded as a suitable candidate for antibacterial drug development; as such, Bacillus anthracis NaMNAT (BA NaMNAT) was heterologously expressed in Escherichia coli for the purpose of inhibitor discovery and crystallography. The crystal structure of BA NaMNAT was determined by molecular replacement, revealing two dimers per asymmetric unit, and was refined to an R factor and R(free) of 0.228 and 0.263, respectively, at 2.3 A resolution. The structure is very similar to that of B. subtilis NaMNAT (BS NaMNAT), which is also a dimer, and another independently solved structure of BA NaMNAT recently released from the PDB along with two ligated forms. Comparison of these and other less related bacterial NaMNAT structures support the presence of considerable conformational heterogeneity and flexibility in three loops surrounding the substrate-binding area.

Crystal structure of Plasmodium falciparum phosphoglycerate kinase: Evidence for anion binding in the basic patch

3-Phosphoglycerate kinase (EC 2.7.2.3) is a key enzyme in the glycolytic pathway and catalyzes an important phosphorylation step leading to the production of ATP. The crystal structure of Plasmodium falciparum phosphoglycerate kinase (PfPGK) in the open conformation is presented in two different groups, namely I222 and P6(1)22. The structure in I222 space group is solved using MAD and refined at 3Å whereas that in P6(1)22A is solved using MR and refined at 2.7Å. I222 form has three monomers in asymmetric unit whereas P6(1)22 form has two monomers in the asymmetric unit. In both crystal forms a sulphate ion is located at the active site where ATP binds, but no Mg(2+) ion is observed. For the first time another sulphate ion is found at the basic patch where the 3-phosphate of 1,3-biphosphoglycerate normally binds. This was found in both chains of P6(1)22 form but only in chain A of I222 form.

The international space station X-ray crystallography facility

Abstract This paper presents an overview of the X-ray Crystallography Facility (XCF) currently under development by UAB/CBSE with major support from three contractors. The XCF is to be used on the International Space Station (ISS) for analysis of macromolecular protein crystals grown on the ISS. A brief summary of protein crystal growth experience on the Space Shuttle Orbiter over nearly 15 years is presented, followed by an explanation of reasons for and advantages of a crystal analysis laboratory on the ISS to support protein crystal growth on that space vehicle. The major elements (called prime items) of the XCF are then described, with emphasis on the critical technologies which have been invented or adapted for the ISS and which could also have scientific and commercial applications outside the ISS arena. The crystal preparation prime item is the first of these major elements. The crystal preparation prime item harvests, prepares, and cryogenically preserves crystals by telerobotic techniques completely controlled by crystallographers on the ground. The X-ray diffraction prime item is the second of these major elements. An advanced X-ray source has been invented and developed for this single-crystal diffraction application. This source is described, together with an advanced goniometer and X-ray detector. The third of these major elements is the command, control, and data prime item which implements command, control and data acquisition from remote ground laboratories, as well as by the ISS crew. A realistic operational scenario is postulated for the XCF on the ISS. This scenario has been analyzed in detail to show that the resources required for the XCF operation are well within the bounds of resources to be available on the ISS. A full rack integrated prototype has been constructed, tested, and evaluated and demonstrates full feasibility of the XCF for the ISS application.