Refined structure of purine nucleoside phosphorylase at 2.75 A resolution.

Human purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of ribonucleosides and 2'-deoxyribonucleosides to the free base and (2'-deoxy)ribose-l-phosphate. The crystal structure previously determined at 3.2 A resolution by multiple isomorphous replacement methods [Ealick, Rule, Carter, Greenhough, Babu, Cook, Habash, Helliwell, Stoeckler, Parks, Chen & Bugg (1990). J. Biol. Chem. 265, 1812-1820] has now been refined at 2.75 A. One important solvent molecule in the active site is found to be hydrogen bonded to Thr242 and Asn243, a second molecule to the Glu210 side chain (rotated out of the substrate-binding pocket), and a third bridges the hydroxyl of Tyr88 and SO(4)(290), located in the phosphate-binding subsite. Hydrophobic interactions dominate the structure and many secondary structural elements are held together by hydrophobic clusters. In the low-resolution structure, the active-site residue Lys244 was modeled to be pointing into the active site, and the refined structure revealed that it is pointing away from the active site. Refinement improved the density for residues 244-249; however, loop 250-263 still shows significant disorder in the native structure. Comparison between crystal structures of native and an inhibitor (THDZ) complex reveals that this flexible loop 250-263 is stabilized by the hydrophobic interactions with the bound inhibitor. The refined structure of PNP is structurally homologous to carboxypeptidase A(CPA), an enzyme which cleaves C-terminus peptides in protein degradation. Similarities and differences between the structures of PNP and CPA are discussed.

Structure-based design of inhibitors of purine nucleoside phosphorylase.

Inhibitors of purine nucleoside phosphorylase may have therapeutic value in the treatment of T-cell proliferative diseases such as T-cell leukemia, in the suppression of host-versus-graft response in organ transplants, and in the treatment of T-cell-mediated autoimmune diseases. Competitive inhibitors of this enzyme have been designed using the three-dimensional structure of the enzyme determined by X-ray crystallography. This approach has resulted in the synthesis of the most potent and membrane-permeable inhibitors of purine nucleoside phosphorylase reported so far.

Comparison of homology models with the experimental structure of a novel serine protease.

A model structure of the human complement enzyme factor D was built based on homology with related serine proteases. A molecular-replacement solution of the factor D crystal structure employing the homology model refined without manual intervention to an R factor of 0.249 with 2.4 A native diffraction data. A multiple isomorphous replacement (MIR) electron-density map was subsequently produced, leading to a model refined at 2.0 A resolution to an R factor of 0.188. A homology model built with commercial modeling software was subjected to the same procedure. Comparisons of the homology models with the final refined MIR structure are presented. Major discrepancies were found in critical active-site regions.

Error detection in crystallographic models.

A variety of criteria were tested for identifying errors in protein crystal coordinates. Statistical analysis was based on comparisons of a highly refined crystal structure and several preliminary models derived from molecular replacement. A protocol employing temperature factors, real-space fit residuals, geometric strains, dihedral angles and shifts from the previous refinement cycle is developed. These results are generally applicable to the detection of errors in partially refined protein crystal structures.

Protein crystal growth in microgravity-temperature induced large scale crystallization of insulin.

One of the major stumbling blocks that prevents rapid structure determination using x-ray crystallography is macromolecular crystal growth. There are many examples where crystallization takes longer than structure determination. In some cases, it is impossible to grow useful crystals on earth. Recent experiments conducted in conjunction with NASA on various Space Shuttle missions have demonstrated that protein crystals often grow larger and display better internal molecular order than their earth-grown counterparts. This paper reports results from three Shuttle flights using the Protein Crystallization Facility (PCF). The PCF hardware produced large, high-quality insulin crystals by using a temperature change as the sole means to affect protein solubility and thus, crystallization. The facility consists of cylinders/containers with volumes of 500, 200, 100, and 50 ml. Data from the three Shuttle flights demonstrated that larger, higher resolution crystals (as evidenced by x-ray diffraction data) were obtained from the microgravity experiments when compared to earth-grown crystals.

Structure-based design of inhibitors of purine nucleoside phosphorylase. 4. A study of phosphate mimics.

9-(3,3-Dimethyl-5-phosphonopentyl)guanine was synthesized and found to be a potent inhibitor of purine nucleoside phosphorylase (PNP) (IC50 = 44 nM). A number of other functional end groups were investigated as phosphate mimics attached to the 9-position of guanine by this same alkyl side chain, which provided a sensitive method for the detection of any interaction of these groups with the phosphate binding site of PNP. Both the sulfonic acid (compound 13) and the carboxylic acid (compound 15) end groups interact significantly with the phosphate binding site, but in different ways, as determined by X-ray crystallographic analysis of the complexes. The sulfonic acid of 13, which binds about one-fourth as tightly as the phosphonate 12, binds in the phosphate subsite much like the phosphonic acid. The carboxylic acid, the interaction of which is much weaker, turns away from the center of the phosphate binding site to form hydrogen bonds with Ser 200 and Met 219. Thus, the only phosphate mimics that bind like phosphate itself are themselves highly ionic, probably with limited ability to penetrate cell membranes.

Structure of human factor D. A complement system protein at 2.0 A resolution.

Factor D, an essential enzyme for the activation of the alternative pathway of the complement system, belongs to the serine protease superfamily. The crystal structure of the enzyme was solved by a combination of multiple isomorphous replacement and molecular replacement methods. The present model was refined to an R-factor of 18.8% using 23,681 observed reflections between 7.5 and 2.0 A resolution, with a root-mean-square deviation from standard bond lengths of 0.016 A. The two non-crystallographically related molecules in the triclinic unit cell have distinctive active site conformations. The protein has the general structural fold of a serine protease, but there are several unique amino acid substitutions resulting in significant alterations in the critical loops responsible for catalysis and substrate specificity in serine proteases. Factor D is the first complement serine protease whose three-dimensional structure has been determined.

Structure-based design of inhibitors of purine nucleoside phosphorylase. 3. 9-Arylmethyl derivatives of 9-deazaguanine substituted on the methylene group.

X-ray crystallography and computer-assisted molecular modeling (CAMM) studies aided in the design of a potent series of mammalian purine nucleoside phosphorylase (PNP) inhibitors. Enhanced potency was achieved by designing substituted 9-(arylmethyl)-9-deazaguanine analogs that interact favorably with all three of the binding subsites of the PNP active site, namely the purine binding site, the hydrophobic pocket, and the phosphate binding site. The most potent PNP inhibitor prepared during our investigation, (S)-9-[1-(3-chlorophenyl)-2-carboxyethyl]-9-deazaguanine (18b), was shown to have an IC50 of 6 nM, whereas the corresponding (R)-isomer was 30-fold less potent.

Structure-based design of inhibitors of purine nucleoside phosphorylase. 2. 9-Alicyclic and 9-heteroalicyclic derivatives of 9-deazaguanine.

Alicyclic and heteroalicyclic derivatives of 9-deazaguanine (2-amino-1,5-dihydro-4H-pyrrolo[3,2-d] [pyrimidin-4-one) are, with one exception, potent inhibitors of purine nucleoside phosphorylase (PNP) equaling the corresponding 9-arylmethyl derivatives previously investigated. The mode of binding of these compounds to PNP was determined by X-ray crystallography.

Structure-based design of inhibitors of purine nucleoside phosphorylase. 1. 9-(arylmethyl) derivatives of 9-deazaguanine.

Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is a salvage enzyme important to the T-cell-mediated part of the immune system and as such is an important therapeutic target. This paper describes the design, synthesis, and enzymatic evaluation of potent, competitive inhibitors of PNP. Potential inhibitors were designed using the three-dimensional structure of the enzyme in an iterative process that involved interactive computer graphics to model the native enzyme and complexes of it with the inhibitors, Monte Carlo-based conformational searching, and energy minimization. Studies of the enzyme/inhibitor complexes were used to determine priorities of the synthetic efforts. The resulting compounds were then evaluated by determination of their IC50 values and by X-ray diffraction analysis using difference Fourier maps. In this manner, we have developed a series of 9-(arylmethyl)-9-deazapurines (2-amino-7-(arylmethyl)-4H-pyrrolo[3,2-d]-pyrimidin-4-ones) that are potent, membrane-permeable inhibitors of the enzyme. The IC50 values of these compounds range from 17 to 270 nM (in 1 mM phosphate), with 9-(3,4-dichlorobenzyl)-9-deazaguanine being the most potent inhibitor. X-ray analysis explained the role of the aryl groups and revealed the rearrangement of hydrogen bonds in the binding of the 9-deazaguanines in the active site of PNP relative to the binding of the 8-aminoguanines that results in more potent inhibition of the enzyme.

Crystal structure of recombinant human interleukin-4.

The crystal structure of recombinant human interleukin-4 (rhuIL-4) was initially determined at 3.5-A resolution by multiple isomorphous replacement techniques and subsequently refined to a resolution of 2.35 A by simulated annealing. The final crystallographic R-factor, based on all data in the range 6.0-2.35 A (7470 reflections), is 0.232. Bond lengths and bond angles in the molecule have root mean square deviations from ideal values of 0.016 A and 2.4 degrees, respectively. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhuIL-4 is a four alpha-helix bundle, which composes approximately 58% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within these connections is a two-stranded antiparallel beta-sheet. Both the tertiary and secondary structures of rhuIL-4 are similar to those of human granulocyte-macrophage colony-stimulating factor. Critical regions for receptor binding are proposed.

Structure of scorpion toxin variant-3 at 1.2 A resolution.

The crystal structure of the variant-3 protein neurotoxin from the scorpion Centruroides sculpturatus Ewing has been refined at 1.2 A resolution using restrained least-squares. The final model includes 492 non-hydrogen protein atoms, 453 protein hydrogen atoms, eight 2-methyl-2,4-pentanediol (MPD) solvent atoms, and 125 water oxygen atoms. The variant-3 protein model geometry deviates from ideal bond lengths by 0.024 A and from ideal angles by 3.6 degrees. The crystallographic R-factor for structure factors calculated from the final model is 0.192 for 17,706 unique reflections between 10.0 to 1.2 A. A comparison between the models of the initial 1.8 A and the 1.2 A refinement shows a new arrangement of the previously poorly defined residues 31 to 34. Multiple conformations are observed for four cysteine residues and an MPD oxygen atom. The electron density indicates that disulfide bonds between Cys12 and Cys65 and between Cys29 and Cys48 have two distinct side-chain conformations. A molecule of MPD bridges neighboring protein molecules in the crystal lattice, and both MPD enantiomers are present in the crystal. A total of 125 water molecules per molecule of protein are included in the final model with B-values ranging from 11 to 52 A2 and occupancies from unity down to 0.4. Comparisons between the 1.2 A and 1.8 A models, including the bound water structure and crystal packing contacts, are emphasized.

Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factor.

The crystal structure of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been determined at 2.8 A resolution using multiple isomorphous replacement techniques. There are two molecules in the crystallographic asymmetric unit, which are related by an approximate non-crystallographic 2-fold axis. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhGM-CSF is a four alpha-helix bundle, which represents approximately 42% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within the connections is a two-stranded antiparallel beta-sheet. The tertiary structure of rhGM-CSF has a topology similar to that of porcine growth factor and interferon-beta. Most of the proposed critical regions for receptor binding are located on a continuous surface at one end of the molecule that includes the C terminus.

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

NASA: 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).^ieng

Application of crystallographic and modeling methods in the design of purine nucleoside phosphorylase inhibitors.

Competitive inhibitors of the salvage pathway enzyme purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) have been designed by using the three-dimensional structure of the enzyme as determined by x-ray crystallography. The process was an iterative one that utilized interactive computer graphics, Monte Carlo-based conformational searching, energy minimization, and x-ray crystallography. The proposed compounds were synthesized and tested by an in vitro assay. Among the compounds designed and synthesized are the most potent competitive inhibitors of purine nucleoside phosphorylase thus far reported.

Protein crystal growth in microgravity.

Protein crystallography is a powerful method for determining the three-dimensional structures of biological macromolecules. Although new methods, such as two-dimensional NMR, have demonstrated promise for determining the structures of small proteins and nucleic acids, the complete atomic arrangements within large proteins can only be determined at present using crystallographic techniques. Such crystallographic studies have been of major importance for establishing structure/function relationships that are fundamental to understanding how enzymes, nucleic acids, and other macromolecules function in biological systems. More recently, crystallographic studies of proteins have become of considerable practical interest within the pharmaceutical and biotechnology industries, as promising tools in drug design and in protein engineering.

Crystallization and preliminary X-ray investigation of factor D of human complement.

Human factor D, an essential enzyme of the alternative pathway of complement activation, has been crystallized. Crystals were grown by vapor diffusion using polyethylene glycol 6000 and NaCl as precipitants. The factor D crystals are triclinic and the space group is P1 with unit cell dimensions a = 40.8 A, b = 64.7 A, c = 40.3 A, alpha = 101.0 degrees, beta = 109.7 degrees, gamma = 74.3 degrees. The unit cell contains two molecules of factor D related by a non-crystallographic 2-fold axis. The crystals grow to dimensions of 0.8 mm x 0.5 mm x 0.2 mm within five days, are stable in the X-ray beam and diffract beyond 2.5 A.

Three-dimensional structure of recombinant human interferon-gamma.

The x-ray crystal structure of recombinant human interferon-gamma has been determined with the use of multiple-isomorphous-replacement techniques. Interferon-gamma, which is dimeric in solution, crystallizes with two dimers related by a noncrystallographic twofold axis in the asymmetric unit. The protein is primarily alpha helical, with six helices in each subunit that comprise approximately 62 percent of the structure; there is no beta sheet. The dimeric structure of human interferon-gamma is stabilized by the intertwining of helices across the subunit interface with multiple intersubunit interactions.