A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated “hairpin” structure of α-synuclein.

FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression. Of all the thousand natural shocks that flesh is heir to, one of the most devastating is amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's Disease. This disorder, which comes in both inherited and random forms, is characterized by degeneration of spinal motor neurons, leading to paralysis and death. The cause of the sporadic form is unknown, but new insight has come from studying the genetic variations that lead to the rarer familial forms. One such gene, accounting for 5%–10% of inherited ALS, is FUS/TLS, which encodes a protein that normally lives in the nucleus of the cell and is involved in the life-cycle of messenger RNA (mRNA). ALS-associated mutations in FUS/TLS cause the protein to mislocalize outside the nucleus into stress granules. Understanding the basis for the toxicity of mislocalized FUS/TLS could lead to new approaches to the treatment of ALS. We have made a yeast model for FUS/TLS cellular toxicity that recapitulates the mislocalization, granular accumulation, and cell death. We have exploited the yeast model to obtain information about what part of the protein is required for proper localization and what part is essential for toxicity. We have also identified several human genes that, when over-expressed in yeast, are able to rescue the cell from the toxicity of mislocalized FUS/TLS. These genes all have functions in mRNA quality control, implicating changes in this pathway in the pathology of ALS.

The x-ray crystal structure of the pyridoxal-5′-phosphate (PLP), S -adenosyl- l -methionine (SAM), and [4Fe–4S]-dependent lysine-2,3-aminomutase (LAM) of Clostridium subterminale has been solved to 2.1-Å resolution by single-wavelength anomalous dispersion methods on a l -selenomethionine-substituted complex of LAM with [4Fe–4S] ²⁺ , PLP, SAM, and l -α-lysine, a very close analog of the active Michaelis complex. The unit cell contains a dimer of hydrogen-bonded, domain-swapped dimers, the subunits of which adopt a fold that contains all three cofactors in a central channel defined by six β/α structural units. Zinc coordination links the domain-swapped dimers. In each subunit, the solvent face of the channel is occluded by an N-terminal helical domain, with the opposite end of the channel packed against the domain-swapped subunit. Hydrogen-bonded ionic contacts hold the external aldimine of PLP and l -α-lysine in position for abstraction of the 3-pro- R hydrogen of lysine by C5′ of SAM. The structure of the SAM/[4Fe–4S] complex confirms and extends conclusions from spectroscopic studies of LAM and shows selenium in Se-adenosyl- l -selenomethionine poised to ligate the unique iron in the [4Fe–4S] cluster upon electron transfer and radical formation. The chain fold in the central domain is in part analogous to other radical–SAM enzymes.

The three-dimensional structure of the N -acyl- l -homoserine lactone hydrolase (AHL lactonase) from Bacillus thuringiensis has been determined, by using single-wavelength anomalous dispersion (SAD) phasing, to 1.6-Å resolution. AHLs are produced by many Gram-negative bacteria as signaling molecules used in quorum-sensing pathways that indirectly sense cell density and regulate communal behavior. Because of their importance in pathogenicity, quorum-sensing pathways have been suggested as potential targets for the development of novel therapeutics. Quorum-sensing can be disrupted by enzymes evolved to degrade these lactones, such as AHL lactonases. These enzymes are members of the metallo-β-lactamase superfamily and contain two zinc ions in their active sites. The zinc ions are coordinated to a number of ligands, including a single oxygen of a bridging carboxylate and a bridging water/hydroxide ion, thought to be the nucleophile that hydrolyzes the AHLs to ring-opened products, which can no longer act as quorum signals.