Betulinic acid induces a novel cell death pathway that depends on cardiolipin modification.

Cancer is associated with strong changes in lipid metabolism. For instance, normal cells take up fatty acids (FAs) from the circulation, while tumour cells generate their own and become dependent on de novo FA synthesis, which could provide a vulnerability to target tumour cells. Betulinic acid (BetA) is a natural compound that selectively kills tumour cells through an ill-defined mechanism that is independent of BAX and BAK, but depends on mitochondrial permeability transition-pore opening. Here we unravel this pathway and show that BetA inhibits the activity of steroyl-CoA-desaturase (SCD-1). This enzyme is overexpressed in tumour cells and critically important for cells that utilize de novo FA synthesis as it converts newly synthesized saturated FAs to unsaturated FAs. Intriguingly, we find that inhibition of SCD-1 by BetA or, alternatively, with a specific SCD-1 inhibitor directly and rapidly impacts on the saturation level of cardiolipin (CL), a mitochondrial lipid that has important structural and metabolic functions and at the same time regulates mitochondria-dependent cell death. As a result of the enhanced CL saturation mitochondria of cancer cells, but not normal cells that do not depend on de novo FA synthesis, undergo ultrastructural changes, release cytochrome c and quickly induce cell death. Importantly, addition of unsaturated FAs circumvented the need for SCD-1 activity and thereby prevented BetA-induced CL saturation and subsequent cytotoxicity, supporting the importance of this novel pathway in the cytotoxicity induced by BetA.

Joint refinement as a tool for thorough comparison between NMR and X-ray data and structures of HU protein.

Joint refinement, i.e., the simultaneous refinement of a structure against both nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic data, was performed on the HU protein from Bacillus stearothermophilus (HUBst). The procedure was aimed at investigating the compatibility of the two data sets and at identifying conflicting information. Wherever important differences were found, such as peptide flips in the main-chain conformation, the data were further analyzed to find the cause. The NMR data showed some errors arising either from the manual interpretation of the spectra or from the incorrect account for spin diffusion. The most important artefact inherent to the X-ray data is the crystal packing of the molecules: the effects range from the limitation of the freedom of the flexible parts of the HUBst molecule to possibly one of the peptide flips.

Specific chemical and structural damage to proteins produced by synchrotron radiation.

Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.

Completeness of NOEs in protein structure: a statistical analysis of NMR.

The completeness of experimentally observed NOE restraints of a set of 97 NMR protein structures deposited in the PDB has been assessed. Completeness is defined as the ratio of the number of experimentally observed NOEs and the number of 'expected NOEs'. A practical definition of 'expected NOEs' based on inter-proton distances in the structures up to a given cut-off distance is proposed. The average completeness for the set of 97 structures is 68, 48, and 26% up to 3, 4, and 5 A cut-off distances, respectively. For recent state-of-the-art structures these numbers are approximately 90, 75, and 45%. Almost 20% of the observed NOEs are between atoms that are further than 5 A apart in the final structures. The completeness is independent of the relative surface accessibility and does not depend strongly on residue type, secondary structure or local precision, although the number of observed NOEs in these classes varies considerably. The completeness of NOE restraints is a useful quality criterion in the course of structure refinement. The completeness per residue is more informative than the number of NOEs per residue, which makes it a useful tool to assess the quality of the NMR data set in relation to the resulting structures.

Validation of nuclear magnetic resonance structures of proteins and nucleic acids: hydrogen geometry and nomenclature.

A statistical analysis is reported of 1,200 of the 1,404 nuclear magnetic resonance (NMR)-derived protein and nucleic acid structures deposited in the Protein Data Bank (PDB) before 1999. Excluded from this analysis were the entries not yet fully validated by the PDB and the more than 100 entries that contained < 95% of the expected hydrogens. The aim was to assess the geometry of the hydrogens in the remaining structures and to provide a check on their nomenclature. Deviations in bond lengths, bond angles, improper dihedral angles, and planarity with respect to estimated values were checked. More than 100 entries showed anomalous protonation states for some of their amino acids. Approximately 250,000 (1.7%) atom names differed from the consensus PDB nomenclature. Most of the inconsistencies are due to swapped prochiral labeling. Large deviations from the expected geometry exist for a considerable number of entries, many of which are average structures. The most common causes for these deviations seem to be poor minimization of average structures and an improper balance between force-field constraints for experimental and holonomic data. Some specific geometric outliers are related to the refinement programs used. A number of recommendations for biomolecular databases, modeling programs, and authors submitting biomolecular structures are given.

Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level.

Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 A resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine. Phosphorylation with DFP caused an unexpected movement in the main chain of a loop that includes residues F288 and F290 of the TcAChE acyl pocket. This is the first major conformational change reported in the active site of any AChE-ligand complex, and it offers a structural explanation for the substrate selectivity of AChE.

Static Laue diffraction studies on acetylcholinesterase.

Acetylcholinesterase (AChE) is one of nature's fastest enzymes, despite the fact that its three-dimensional structure reveals its active site to be deeply sequestered within the molecule. This raises questions with respect to traffic of substrate to, and products from, the active site, which may be investigated by time-resolved crystallography. In order to address one aspect of the feasibility of performing time-resolved studies on AChE, a data set has been collected using the Laue technique on a trigonal crystal of Torpedo californica AChE soaked with the reversible inhibitor edrophonium, using a total X-ray exposure time of 24 ms. Electron-density maps obtained from the Laue data, which are of surprisingly good quality compared with similar maps from monochromatic data, show essentially the same features. They clearly reveal the bound ligand, as well as a structural change in the conformation of the active-site Ser200 induced upon binding.

Structure of acetylcholinesterase complexed with the nootropic alkaloid, (-)-huperzine A.

(-)-Huperzine A (HupA) is found in an extract from a club moss that has been used for centuries in Chinese folk medicine. Its action has been attributed to its ability to strongly inhibit acetylcholinesterase (AChE). The crystal structure of the complex of AChE with optically pure HupA at 2.5 A resolution shows an unexpected orientation for the inhibitor with surprisingly few strong direct interactions with protein residues to explain its high affinity. This structure is compared to the native structure of AChE devoid of any inhibitor as determined to the same resolution. An analysis of the affinities of structural analogues of HupA, correlated with their interactions with the protein, shows the importance of individual hydrophobic interactions between HupA and aromatic residues in the active-site gorge of AChE.

Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.