Herman A. Schreuder

Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 Å resolution: analysis of the enzyme-substrate and enzyme-product complexes

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)

X-ray structure of antistasin at 1.9 A resolution and its modelled complex with blood coagulation factor Xa.

The three‐dimensional structure of antistasin, a potent inhibitor of blood coagulation factor Xa, from the Mexican leech Haementeria officinalis was determined at 1.9 Å resolution by X‐ray crystallography. The structure reveals a novel protein fold composed of two homologous domains, each resembling the structure of hirustasin, a related 55‐residue protease inhibitor. However, hirustasin has a different overall shape than the individual antistasin domains, it contains four rather than two β‐strands, and does not inhibit factor Xa. The two antistasin domains can be subdivided into two similarly sized subdomains with different relative orientations. Consequently, the domain shapes are different, the N‐terminal domain being wedge‐shaped and the C‐terminal domain flat. Docking studies suggest that differences in domain shape enable the N‐terminal, but not C‐terminal, domain of antistasin to bind and inhibit factor Xa, even though both have a very similar reactive site. Furthermore, a putative exosite binding region could be defined in the N‐terminal domain of antistasin, comprising residues 15‐17, which is likely to interact with a cluster of positively charged residues on the factor Xa surface (Arg222/Lys223/Lys224). This exosite binding region explains the specificity and inhibitory potency of antistasin towards factor Xa. In the C‐terminal domain of antistasin, these exosite interactions are prevented due to the different overall shape of this domain.

Structure and Function of Mutant Arg44Lys of 4‐Hydroxybenzoate Hydroxylase

Arg44, located at the si-face side of the flavin ring in 4–hydroxybenzoate hydroxylase, was changed to lysine by site-specific mutagenesis. Crystals of [R44K]4-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-nm resolution. The structure of [R44K]4-hydroxybenzoate hydroxylase is identical to the wild-type enzyme except for local changes in the vicinity of the mutation. The peptide unit between Ile43 and Lys44 is flipped by about 180° in 50% of the molecules. The φ,ψ angles in both the native and flipped conformation are outside the allowed regions and indicate a strained conformation. [R44K]4-Hydroxybenzoate hydroxylase has a decreased affinity for the flavin prosthetic group. This is ascribed to the lost interactions between the side chain of Arg44 and the diphosphoribose moiety of the FAD. The replacement of Arg44 by Lys does not change the position of the flavin ring which occupies the same interior position as in wild type. [R44K]4-Hydroxybenzoate hydroxylase fully couples flavin reduction to substrate hydroxylation. Stopped-flow kinetics showed that the effector role of 4-hydroxybenzoate is largely conserved in the mutant. Replacement of Arg44 by Lys however affects NADPH binding, resulting in a low yield of the charge-transfer species between reduced flavin and NADP+. It is inferred from these data that Arg44 is indispensable for optimal catalysis.

Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase. Implications for NADPH recognition and structural stability.

Phe161 and Arg166 of p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe161 and Arg166 were selectively changed by site‐directed mutagenesis. F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 Å is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 Å revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH.

The transfer of protein crystals from their original mother liquor to a solution with a completely different precipitant

A procedure is described for the transfer of protein crystals from an ammonium sulfate-containing mother liquor to a solution with another precipitant, such as polyethylene glycol. The suitable concentration of the alternative precipitant is established via a novel protocol, using a hanging-drop equilibration method. This crystal transfer procedure is illustrated by experiments with crystals of trypanosomal triosephosphate isomerase and bacterial p-hydroxybenzoate hydroxylase, but it might have more general applicability.

Protein Crystallography and Drug Discovery

Publisher Summary The impact of structural biology on the daily work of medicinal chemists has been particularly strong. Structural information not only clarifies structure–activity relationships, reveals binding modes and bioactive conformations, and unveils new binding pockets or allosteric binding sites; it also opens new and diverse drug discovery avenues, such as in silico screening, the rational design of focused chemical libraries, and the de novo design of new ligand scaffolds. X-ray crystallography has played a major role in the structural biology revolution, as the most powerful technique to decipher the 3D architecture of biological macromolecules at atomic or near atomic resolution. Biological crystallography can be applied to any macromolecular targets or assemblies, irrespective of their size and complexity, provided that crystals of sufficient quality can be produced. This chapter describes how crystallographic data can contribute today to the different phases of pharmaceutical research. It emphasizes the strengths but also the technical limitations of protein crystallography, so that any medicinal chemist engaging in a new research program and having access to a structural biology group can gauge if, and how, his project could potentially benefit from this technology. A brief outline of the basic principles and methods of protein crystallography is provided. Medicinal chemists, in particular those working in the industry, have access to large, public as well as proprietary, depositories of refined crystal structures. This study would contribute to a more effective communication between chemists and their fellow crystallographers.

DETERMINATION OF BETA-GALACTOSIDASE ACTIVITY IN THE INTESTINAL-TRACT OF MICE BY ION-EXCHANGE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING EPSILON-N-1-(1-DEOXYLACTULOSYL)-L-LYSINE AS SUBSTRATE

epsilon-N-1-(1-Deoxylactulosyl)-L-lysine was synthesized and used as a substrate to assay beta-galactosidase activity. epsilon-N-1-(1-Deoxylactulosyl)-L-lysine and its degradation product epsilon-N-1-(1-deoxyfructosyl)-L-lysine were detected by high-voltage paper electrophoresis and ion-exchange high-performance liquid chromatography. The beta-galactosidase activity in different parts of the intestinal tract of germ-free and control mice was determined and compared with a beta-galactosidase activity which degrades lactose at pH 8.5 and 5.0 and which corresponded with bacterial and host enzymatic activities, respectively.

Application of conformationally restricted peptidomimetics to modeling the bound conformation of peptide antagonists with the IL-1 receptor

A collection of complementary peptide caricatures that closely mimic low-energy (presumably highly populated) conformations of amino acids of interest would constitute a valuable tool set to study the interactions of small peptide ligands with their biological targets. Our general strategy for the design, synthesis and application of peptidomimetics is presented. An illustration of how structural information from mimetics combined with cutting edge biophysical data can be used to derive a model for the bound conformation of an 11-mer peptide antagonist with the IL-1 receptor is given.

Chapter 22 – Protein Crystallography and Drug Discovery

Protein crystallography is an integral part of pharmaceutical research. On-going developments in miniaturization, robotics, etc. have greatly increased the chance of obtaining crystal structures of the target of interest. Often, crystal structures will already be available from the public domain at the start of a project. Here we discuss all aspects of protein crystallography within drug discovery, starting with a historic overview, followed by the crystallization of proteins, with emphasis on requirements for crystallizable proteins and the preparation of protein–ligand complexes. Also, specific issues and tricks for protein families like proteases, kinases, and G-protein-coupled receptors are discussed. The theoretical background of crystallography and the effect of disorder are briefly discussed, so that medicinal chemists and modelers gain a better understanding of the strengths and limitations of protein crystallography. The second part discusses the application of protein crystallography in the drug discovery value chain, especially fragment-based screening, hit selection, and lead optimization, where historically crystallography had its largest impact.

IL-1 antagonist discovery

Interleukin 1 (IL-1) is a pivotal pro-inflammatory cytokine involved in many types of inflammatory and autoimmune diseases [1]. IL-1 production whether initiated by infection or host antigen, initiates an inflammatory cascade which includes the induction of other mediators (e.g. IL-6, IL-8), upregulation of adhesion molecules (e.g. ICAM-1, E-selectin) and potent synergistic activity with other macrophage, proinflammatory cytokines (e.g. tumor necrosis factor) [2,3]. The IL-1 family consists of three structurally related naturally occurring ligands: two agonists, IL-1 α, and IL-1s; and one antagonist, the IL-1 receptor antagonist (IL-1RA) [4-6]. IL-1 is the only cytokine known to have a naturally occurring receptor antagonist. Two distinct cell membrane receptors have been characterized: The type I, IL-1 receptor (IL1RtI) responsible for biological responses; and the type II receptor (IL-1RtII), which is shed, and acts as a decoy to modulate IL-1 activity [7-11]. The existence of a naturally occurring antagonist and a decoy receptor further illustrates the importance and need for the tight regulation of IL-1 activity and its critical role in inflammation.