Keith D. Watenpaugh

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.

Macromolecular crystallography at cryogenic temperatures: Current Opinion in Structural Biology 1991, 1: 1012–1015

Abstract Cryocrystallography of biological macromolecules is an underused technique for improving the quality of crystallographically determined structures. Although the technique may not be useful in all cases, a growing number of examples show that radiation damage, protein instability or thermal motion can be greatly reduced by collecting X-ray diffraction data at cryogenic temperatures. Most macromolecular crystals can be cooled rapidly and diffract at least as well as they do at room temperature, although showing increased mosaic character.

Non-peptidic HIV protease inhibitors: C2-symmetry-based design of bis-sulfonamide dihydropyrones

Potent, non-peptidic, dihydropyrone sulfonamide HIV protease inhibitors have been previously described. Crystallographic analysis of dihydropyrone sulfonamide inhibitor/HIV protease complexes suggested incorporation of a second, C2 symmetry-related sulfonamide group. Selected bis-sulfonamide dihydropyrone analogues display high HIV protease inhibitory activity.

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.

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.

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.