X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif.

Junctional adhesion molecules (JAMs) are a family of immunoglobulin-like single-span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte trans migration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 A resolution. rsJAM consists of two immunoglobulin-like domains that are connected by a conformationally restrained short linker. Two rsJAM molecules form a U-shaped dimer with highly complementary interactions between the N-terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U-shaped JAM dimers are oriented in cis on the cell surface and form a two-dimensional network by trans-interactions of their N-terminal domains with JAM dimers from an opposite cell surface.

Piperidine renin inhibitors: from leads to drug candidates.

Non-peptidomimetic renin inhibitors of the piperidine type represent a novel structural class of compounds potentially free of the drawbacks seen with peptidomimetic compounds so far. Synthetic optimization in two structural series focusing on improvement of potency, as well as on physicochemical properties and metabolic stability, has led to the identification of two candidate compounds 14 and 23. Both display potent and long-lasting blood pressure lowering effects in conscious sodium-depleted marmoset monkeys and double transgenic rats harboring both the human angiotensinogen and the human renin genes. In addition, 14 normalizes albuminuria and kidney tissue damage in these rats when given over a period of 4 weeks. These data suggest that treatment of chronic renal failure patients with a renin inhibitor might result in a significant improvement of the disease status.

Crystal structures of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase, GlmU, in apo form at 2.33 A resolution and in complex with UDP-N-acetylglucosamine and Mg(2+) at 1.96 A resolution.

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential bacterial enzyme with both an acetyltransferase and a uridyltransferase activity which have been mapped to the C-terminal and N-terminal domains, respectively. GlmU performs the last two steps in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), which is an essential precursor in both the peptidoglycan and the lipopolysaccharide metabolic pathways. GlmU is therefore an attractive target for potential antibiotics. Knowledge of its three-dimensional structure would provide a basis for rational drug design. We have determined the crystal structures of Streptococcus pneumoniae GlmU (SpGlmU) in apo form at 2.33 A resolution, and in complex with UDP-N-acetyl glucosamine and the essential co-factor Mg(2+) at 1.96 A resolution. The protein structure consists of an N-terminal domain with an alpha/beta-fold, containing the uridyltransferase active site, and a C-terminal domain with a long left-handed beta-sheet helix (LbetaH) domain. An insertion loop containing the highly conserved sequence motif Asn-Tyr-Asp-Gly protrudes from the left-handed beta-sheet helix domain. In the crystal, S. pneumoniae GlmU forms exact trimers, mainly through contacts between left-handed beta-sheet helix domains. UDP-N-acetylglucosamine and Mg(2+) are bound at the uridyltransferase active site, which is in a closed form. We propose a uridyltransferase mechanism in which the activation energy of the double negatively charged phosphorane transition state is lowered by charge compensation of Mg(2+) and the side-chain of Lys22.

Observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex.

BACKGROUND: Molecular interactions among cytokines and cytokine receptors form the basis of many cell-signaling pathways relevant to immune function. Interferon-gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different but structurally related transmembrane chains: the high-affinity receptor-binding subunit (IFN-gammaRalpha) and a species-specific accessory factor (AF-1 or IFN-gammaRbeta). In the signaling complex, the two receptors probably interact with one another through their extracellular domains. Understanding the atomic interactions of signaling complexes enhances the ability to control and alter cell signaling and also provides a greater understanding of basic biochemical processes. RESULTS: The crystal structure of the complex of human IFN-gamma with the soluble, glycosylated extracellular part of IFN-gammaRalpha has been determined at 2.9 A resolution using multiwavelength anomalous diffraction methods. In addition to the expected 2:1 complex, the crystal structure reveals the presence of a third receptor molecule not directly associated with the IFN-gamma dimer. Two distinct intermolecular contacts, involving the edge strands of the C-terminal domains, are observed between this extra receptor and the 2:1 receptor-ligand complex thereby forming a 3:1 complex. CONCLUSIONS: The observed interactions in the 2:1 complex of the high-affinity cell-surface receptor with the IFN-gamma cytokine are similar to those seen in a previously reported structure where the receptor chains were not glycosylated. The formation of beta-sheet packing interactions between pairs of IFN-gammaRalpha receptors in these crystals suggests a possible model for receptor oligomerization of Ralpha and the structurally homologous Rbeta receptors in the fully active IFN-gamma signaling complex.

Purification and crystallization of the extracellular domain of human neutral endopeptidase (neprilysin) expressed in Pichia pastoris.

Neutral endopeptidase (NEP) is a mammalian zinc metalloprotease involved in the inactivation of a wide variety of regulatory peptides such as enkephalins and atrial natiuretic factor. The soluble extracellular domain of NEP (sNEP) was expressed in the methylotrophic yeast Pichia pastoris. The protein was purified to homogeneity and single crystals have been obtained. Enzymatic deglycosylation of the enzyme was essential for the production of crystals suitable for X-ray analysis for both the NEP-phosphoramidon binary complex and the apo enzyme.

Structure of human neutral endopeptidase (Neprilysin) complexed with phosphoramidon.

Neutral endopeptidase is a mammalian type II integral membrane zinc-containing endopeptidase, which degrades and inactivates a number of bioactive peptides. The range of substrates cleaved by neutral endopeptidase in vitro includes the enkephalins, substance P, endothelin, bradykinin and atrial natriuretic factor. Due to the physiological importance of neutral endopeptidase in the modulation of nociceptive and pressor responses there is considerable interest in inhibitors of this enzyme as novel analgesics and anti-hypertensive agents. Here we describe the crystal structure of the extracellular domain (residues 52-749) of human NEP complexed with the generic metalloproteinase inhibitor phosphoramidon at 2.1 A resolution. The structure reveals two multiply connected folding domains which embrace a large central cavity containing the active site. The inhibitor is bound to one side of this cavity and its binding mode provides a detailed understanding of the ligand-binding and specificity determinants.

From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase.

Naturally-occurring phytases having the required level of thermostability for application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequence comparisons. A consensus enzyme based on 13 fungal phytase sequences had normal catalytic properties, but showed an unexpected 15-22 degrees C increase in unfolding temperature compared with each of its parents. As a first step towards understanding the molecular basis of increased heat resistance, the crystal structure of consensus phytase was determined and compared with that of Aspergillus niger phytase. Aspergillus niger phytase unfolds at much lower temperatures. In most cases, consensus residues were indeed expected, based on comparisons of both three-dimensional structures, to contribute more to phytase stabilization than non-consensus amino acids. For some consensus amino acids, predicted by structural comparisons to destabilize the protein, mutational analysis was performed. Interestingly, these consensus residues in fact increased the unfolding temperature of the consensus phytase. In summary, for fungal phytases apparently an unexpected direct link between protein sequence conservation and protein stability exists.

Crystal engineering: a case study using the 24 kDa fragment of the DNA gyrase B subunit from Escherichia coli.

Site-directed mutagenesis was used to determine the efficacy of changing surface residues to improve crystal quality. Nine mutants of the 24 kDa fragment of the Escherichia coli DNA gyrase B subunit were produced, changing residues on the protein's surface. The mutations changed either the charge or the polarity of the wild-type amino acid. It was found that single amino-acid changes on the surface could have a dramatic effect on the crystallization properties of the protein and generally resulted in an improvement in the number of crystal-screen hits as well as an improvement in crystal quality. It is concluded that crystal engineering is a valuable tool for protein crystallography.

Crystal engineering: deletion mutagenesis of the 24 kDa fragment of the DNA gyrase B subunit from Staphylococcus aureus.

The 24 kDa fragment of DNA gyrase B from Staphylococcus aureus was expressed in Escherichia coli and purified for crystallization. Crystals of the wild-type protein grew in the presence of cyclothialidine but proved difficult to reproduce. In order to improve the crystallization, the flexible regions of the protein were deleted by mutagenesis. The mutant proteins were analyzed by differential scanning calorimetry and the most stable mutants produced crystals. It was possible to reproducibly grow single well defined crystals in the microbatch system which belonged to the space group C2 and diffracted isotropically to approximately 2 A resolution.

Crystal structure of Aspergillus niger pH 2.5 acid phosphatase at 2. 4 A resolution.

The crystal structure of Aspergillus niger pH 2.5 acid phosphatase (EC 3.1.3.2) has been determined at 2.4 A resolution. In the crystal, two dimers form a tetramer in which the active sites are easily accessible to substrates. The main contacts in the dimer come from the N termini, each lying on the surface of the neighbouring molecule. The monomer consists of two domains, with the active site located at their interface. The active site has a highly conserved catalytic center and a charge distribution, which explains the highly acidic pH optimum and the broad substrate specificity of the enzyme.

Crystallization and preliminary crystallographic analysis of an NADH oxidase that functions in peroxide reduction in Thermus aquaticus YT-1.

NADH oxidase from Thermus aquaticus is a thermostable flavoenzyme that is similar in amino-acid sequence and other properties to the flavoenzyme component of the NADH peroxidase systems from Salmonella typhimurium and Amphibacillus xylanus. The enzyme has been isolated from T. aquaticus and crystallized using the hanging-drop method of vapour diffusion with sodium citrate as a precipitant at pH 8.5. The crystals belong to the hexagonal space group P622 with unit-cell dimensions a = b = 89.9, c = 491.6 A, and diffract to 2.5 A resolution.

Renin inhibition by substituted piperidines: a novel paradigm for the inhibition of monomeric aspartic proteinases?

BACKGROUND: The aspartic proteinase renin catalyses the first and rate-limiting step in the conversion of angiotensinogen to the hormone angiotensin II, and therefore plays an important physiological role in the regulation of blood pressure. Numerous potent peptidomimetic inhibitors of this important drug target have been developed, but none of these compounds have progressed past clinical phase II trials. Limited oral bioavailability or excessive production costs have prevented these inhibitors from becoming new antihypertensive drugs. We were interested in developing new nonpeptidomimetic renin inhibitors. RESULTS: High-throughput screening of the Roche compound library identified a simple 3, 4-disubstituted piperidine lead compound. We determined the crystal structures of recombinant human renin complexed with two representatives of this new class. Binding of these substituted piperidine derivatives is accompanied by major induced-fit adaptations around the enzyme's active site. CONCLUSIONS: The efficient optimisation of the piperidine inhibitors was facilitated by structural analysis of the renin active site in two renin-inhibitor complexes (some of the piperidine derivatives have picomolar affinities for renin). These structural changes provide the basis for a novel paradigm for inhibition of monomeric aspartic proteinases.

The structure and function of the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Haemophilus influenzae.

The gene encoding the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase of Haemophilus influenzae has been cloned and expressed in Escherichia coli. A complex of the purified protein with a substrate analog has been crystallized and its structure solved by multiple anomalous dispersion using phase information obtained from a single crystal of selenomethione-labeled protein. The enzyme folds into a four-stranded antiparallel beta-sheet flanked on one side by two alpha-helices and on the other by three consecutive alpha-helices, giving a novel beta1alpha1beta2beta3alpha2beta4alpha3alpha4alpha5 polypeptide topology. The three-dimensional structure of a binary complex has been refined at 2.1 A resolution. The location of the substrate analog and a sulfate ion gives important insight into the molecular mechanism of the enzyme.

Crystal structure and reaction mechanism of 7,8-dihydroneopterin aldolase from Staphylococcus aureus.

Dihydroneopterin aldolase catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin during the de novo synthesis of folic acid from guanosine triphosphate. The gene encoding the dihydroneopterin aldolase from S. aureus has been cloned, sequenced and expressed in Escherichia coli. The protein has been purified for biochemical characterization and its X-ray structure determined at 1.65 A resolution. The protein forms an octamer of 110,000 Mr molecular weight. Four molecules assemble into a ring, and two rings come together to give a cylinder with a hole of at least 13 A diameter. The structure of the binary complex with the product 6-hydroxymethyl-7,8-dihydropterin has defined the location of the active site. The structural information and results of site directed mutagenesis allow an enzyme reaction mechanism to be proposed.

Structure and function of the dihydropteroate synthase from Staphylococcus aureus.

The gene encoding the dihydropteroate synthase of staphylococcus aureus has been cloned, sequenced and expressed in Escherichia coli. The protein has been purified for biochemical characterization and X-ray crystallographic studies. The enzyme is a dimer in solution, has a steady state kinetic mechanism that suggests random binding of the two substrates and half-site reactivity. The crystal structure of apo-enzyme and a binary complex with the substrate analogue hydroxymethylpterin pyrophosphate were determined at 2.2 A and 2.4 A resolution, respectively. The enzyme belongs to the group of "TIM-barrel" proteins and crystallizes as a non-crystallographic dimer. Only one molecule of the substrate analogue bound per dimer in the crystal. Sequencing of nine sulfonamide-resistant clinical isolates has shown that as many as 14 residues could be involved in resistance development. The residues are distributed over the surface of the protein, which defies a simple interpretation of their roles in resistance. Nevertheless, the three-dimensional structure of the substrate analogue binary complex could give important insight into the molecular mechanism of this enzyme.

Crystal structure of phytase from Aspergillus ficuum at 2.5 A resolution.

Phytase is a high molecular weight acid phosphatase. The structure has an alpha/beta-domain similar to that of rat acid phosphatase and an alpha-domain with a new fold.

A single amino acid substitution in Staphylococcus aureus dihydrofolate reductase determines trimethoprim resistance.

A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate reductase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution was found in all S. aureus TMP-resistant clinical isolates tested. In order to explore the structural role of Tyr98 in TMP-resistance the ternary complexes of the chromosomal S. aureus DHFR (SaDHFR) with methotrexate (MTX) and TMP in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98Y) ternary folate-NADPH complex have been determined by X-ray crystallography. Critical evidence concerning the resistance mechanism has also been provided by NMR spectral analyses of 15N-labelled TMP in the ternary complexes of both wild-type and mutant enzyme. These studies show that the mutation results in loss of a hydrogen bond between the 4-amino group of TMP and the carbonyl oxygen of Leu5. This mechanism of resistance is predominant in both transferable plasmid-encoded and non-transferable chromosomally encoded resistance. Knowledge of the resistance mechanism at a molecular level could help in the design of antibacterials active against multi-resistant Staphylococcus aureus (MRSA), one of todays most serious problems in clinical infectology.

Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases.

Obtaining high quality protein crystals remains a rate-limiting step in the determination of three-dimensional X-ray structures. A frequently encountered problem in this respect is the high or heterogeneous carbohydrate content of many eukaryotic proteins. A number of reports have demonstrated the use of enzymatic deglycosylation in the crystallization of certain glycoproteins. Although this is an attractive tool, there are some problems that hinder the more widespread use of glycosidases in crystallization. First, commercially available glycosidases are relatively expensive, which virtually prohibits their use on a large scale. Second, the glycosidase must be removed from the glycoprotein of interest following deglycosylation, which is not always straightforward. To circumvent these problems we have cloned the two most generally useful glycosidases, peptide-N-glycosidase F and endoglycosidase F1 from Flavobacterium meningosepticum, as fusion proteins with glutathione S-transferase. The fusion not only allows rapid purification of these enzymes from Escherichia coli cell extracts, but also permits rapid removal from target proteins following deglycosylation. We have used these enzymes to obtain crystals of phytase from Aspergillus ficuum and acid phosphatase from Aspergillus niger and to obtain a new crystal form of recombinant human renin.