Howard M. Einspahr

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.

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.

Resolution of microheterogeneity associated with recombinant HIV-1 heterodimeric reverse transcriptase.

HIV-1 reverse transcriptase (RT) has been successfully expressed as a biologically active recombinant protein in Escherichia coli and purified to homogeneity. After partial purification, RT was obtained primarily in a heterodimeric form represented by two subunits of 66 and 51 kDa, but the preparation also included several forms distinguishable in size and charge by chromatography on ionic-exchange and gel-filtration columns. We have developed a purification method that yields a single heterodimeric form of RT. Our strategy involves the selection of RT molecules exhibiting uniformity in elution from QAE Sepharose anion-exchange columns and Superose 12 gel-filtration columns. In the former, RT is resolved into multiple peaks on the basis of enzymatic activity, one of which represents highly active and pure p66:p51 heterodimeric RT. This highly active RT fraction, after gel-filtration chromatography, yields a compositionally pure protein product free of observable microheterogeneity by 1D and 2D polyacrylamide gel electrophoresis under a variety of conditions. Furthermore, the RNAse H enzymatic activity associated with HIV-1 RT has been demonstrated to coelute with the purified polymerase activity during gel filtration at a size (120 kDa) consistent with its location on the heterodimeric protein molecule.

Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum

The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3(1)21 or its enantiomer, with a = b = 131.2(7) A, c = 152.6(9) A, and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (lambda = 0.95 A) to about 3.0 A resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.

Protein crystal growth results from the United States Microgravity Laboratory-1 mission

Protein crystal growth experiments have been performed on fourteen space shuttle missions between April 1985 and June 1992. These space shuttle missions have been used to grow crystals of a variety of proteins using vapour diffusion, liquid diffusion, and temperature induced crystallization techniques. The United States Microgravity Laboratory-1 mission (june 25-July 9, 1992) was a space lab mission dedicated to experiments involved in materials processing. New protein crystal growth hardware was developed to allow in-orbit examination of initial crystal growth results, the knowledge from which was used on subsequent days to prepare new crystal growth experiments. The hardware developed specifically for the USML-1 mission is discussed along with preliminary experimental results.

Soybean trypsin inhibitor. An IL-1-like protein?

Soybean trypsin inhibitor (SBTI) shares some structural homology with interleukin-1 (IL-1) and was tested for IL-1 bioactivity. Human T-cells proliferated maximally when stimulated with PMA and SBTI but failed to respond to either stimulus alone. This response was abrogated by neutralizing antibodies to IL-1 beta but not to IL-1 alpha. However, immunoblots showed no cross-reactivity between SBTI and anti-IL-1 antibodies. Furthermore, SBTI did not bind to IL-1 receptors on YT cells and did not activate a murine T-lymphoma or human T-hybridoma. Supernantants from monocytes stimulated with SBTI contained significant levels of IL-1 activity. The data show that SBTI has no direct IL-1 activity but can stimulate T-cells indirectly through an IL-1 dependent mechanism.

Crystallization of recombinant human interleukin 1β

Abstract The gene for the fully processed form of human interleukin 1β was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in E. coli . The protein produced in E. coli . was purified to homogeneity by standard chromatographic methods, including adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 FPLC column, and anion exchange chromatography on QAE Sepharose. The result is a biologically active protein, rIL-1β, that migrates on two-dimensional gels as a single spot with a pI of 6.5 ± 0.2 and a molecular mass of 17, 500 daltons. Crystals of rIL-1β have been produced from concentrated solutions of the protein by ammonium sulfate precipitation. The crystals are tetragonal, have space group P4 1 or its enantiomer, have lattice constants of a = 58.46(1) A and c = 77.02(3) A, and scatter to at least 2 A resolution. A structure determination ba these crystals is underway.

Protein crystal growth aboard the U.S. space shuttle flights STS-31 and STS-32

The first microgravity protein crystal growth experiments were performed on Spacelab I by Littke and John. These experiments indicated that the space grown crystals, which were obtained using a liquid-liquid diffusion system, were larger than crystals obtained by the same experimental system on earth. Subsequent experiments were performed by other investigators on a series of space shuttle missions from 1985 through 1990. The results from two of these shuttle flights (STS-26 and STS-29) have been described previously. The results from these missions indicated that the microgravity grown crystals for a number of different proteins were larger, displayed more uniform morphologies, and yielded diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth. This paper presents the results obtained from shuttle flight STS-32 (flown in January, 1990) and preliminary results from the most recent shuttle flight, STS-31 (flown in April, 1990).

Exploiting the Molecular Template of Angiotensinogen in the Discovery and Design of Peptidyl, Pseudopeptidyl and Peptidemimetic Inhibitors of Human Renin: A Structure-Activity Perspective

The design of potent and pharmacologically effective, substrate-related inhibitors of renin has been the subject of intensive pharmaceutical discovery research for about one decade. Milestone achievements in synthetic tailoring of fragment analogs of angiotensinogen (ANG; Figure 1) have been documented in terms of identifying renin inhibitors of subnanomolar potency, sustained in vivo hypotensive activity, stability towards proteolytic degradation, and, more recently, oral bioavailability and decreased systemic clearance.1 By chemical modification of ANG-based derivatives, structure-activity analysis, and computer-assisted molecular modeling of peptidyl, pseudopeptidyl and peptidemimetic inhibitors using 3-D structural models of human renin, there currently exists a rather sophisticated wealth of information of relevance to the “rational” design of prototypic renin-targeted cardiovascular therapeutic agents. Such efforts have bridged biochemistry, medicinal chemistry, computational and biophysical chemistry, and in vivo pharmacology including, in a few cases, clinical evaluation in humans.

Crystal forms of a lysine-49 phospholipase A2 from the eastern cottonmouth snake

Abstract As part of an effort to determine the structure of a lysine-49 variant phospholipase A2 from the venom of a North American pit viper, the eastern cottonmouth (Agkistrodon piscivorus piscivorus), we have produced five different crystal forms grown under a variety of crystallization conditions. They include an orthorthombic form (P212121 or P21212; a=87.8(3) A, b=76.2(3) A,c=57.4(4) A), an hexagonal form (space group P6122 or its enantiomer; a=b=62.22(3) A, c=137.1(3) A) and two tetragonal forms - a neutral pH form (space group P41212 or its enantiomer; a = b = 81.99(3) A) and high pH form (space group P41212; a = b = 71.5(1) A, c = 57.6(2) A one molecule per asymmetric unit) — the latter of which was used for structure determination.