Among the many surprises to arise from studies of prion biology, perhaps the most unexpected is the strain phenomenon whereby a single protein can misfold into structurally distinct, infectious states that cause distinguishable phenotypes. Similarly, proteins can adopt a spectrum of conformations in non-infectious diseases of protein folding; some are toxic and others are well tolerated. However, our understanding of the structural differences underlying prion strains and how these differences alter their physiological impact remains limited. Here we use a combination of solution NMR, amide hydrogen/deuterium (H/D) exchange and mutagenesis to study the structural differences between two strain conformations, termed Sc4 and Sc37 (ref. 5), of the yeast Sup35 prion. We find that these two strains have an overlapping amyloid core spanning most of the Gln/Asn-rich first 40 amino acids that is highly protected from H/D exchange and very sensitive to mutation. These features indicate that the cores are composed of tightly packed beta-sheets possibly resembling 'steric zipper' structures revealed by X-ray crystallography of Sup35-derived peptides. The stable structure is greatly expanded in the Sc37 conformation to encompass the first 70 amino acids, revealing why this strain shows increased fibre stability and decreased ability to undergo chaperone-mediated replication. Our findings establish that prion strains involve large-scale conformational differences and provide a structural basis for understanding a broad range of functional studies, including how conformational changes alter the physiological impact of prion strains.

Cytochrome oxidase is a membrane protein complex that catalyzes reduction of molecular oxygen to water and utilizes the free energy of this reaction to generate a transmembrane proton gradient during respiration. The electron entry site in subunit II is a mixed-valence dinuclear copper center in enzymes that oxidize cytochrome c. This center has been lost during the evolution of the quinoloxidizing branch of cytochrome oxidases but can be restored by engineering. Herein we describe the crystal structures of the periplasmic fragment from the wild-type subunit II (CyoA) of Escherichia coli quinol oxidase at 2.5-A resolution and of the mutant with the engineered dinuclear copper center (purple CyoA) at 2.3-A resolution. CyoA is folded as an 11-stranded mostly antiparallel 13-sandwich followed by three a-helices. The dinuclear copper center is located at the loops between strands 185-.16 and 189-1310. The two coppers are at a 2.5-A distance and symmetrically coordinated to the main ligands that are two bridging cysteines and two terminal histidines. The residues that are distinct in cytochrome c and quinol oxidases are around the dinuclear copper center. Structural comparison suggests a common ancestry for subunit II of cytochrome oxidase and blue copper-binding proteins.Two main branches of the cytochrome oxidase family in bacteria alternatively use cytochrome c or quinol as the electron donor (1, 2). They typically contain three subunits that are homologous to the key components of the mitochondrial enzyme. Subunit I is largely buried in the membrane and forms the core of the complex, harboring the heme-copper active site. Subunit II has been predicted to be composed of an N-terminal transmembrane helical hairpin and a hydrophilic C-terminal domain that resides in the outer side of the cytoplasmic membrane (3). In cytochrome c oxidases, this domain contains a copper center known as CUA that functions as the primary acceptor for the electron from cytochrome c (4, 5). Subunit III is a membrane protein without redox centers (3).The CUA site is a single electron acceptor and donor. It has been spectroscopically characterized as a dinuclear center with a mixed valence [Cu(1.5)-Cu(1.5)]S = 1/2 configuration (4, 6, 7), where the coppers must be in very similar chemical environments (8). The key amino acids involved in the binding site are found in the CCHM amino acid motif, which is preceded by an invariant His in all subunit II sequences of cytochrome c oxidases (Fig. 1). None of these residues are conserved in quinol oxidases lacking the metal center (9, 12, 13). The "lost" CUA site has been reengineered to subunit II of the Escherichia coli quinol oxidase (CyoA) by a simultaneous substitution of six residues that results in a protein with purple color (9) (hereafter referred to as purple CyoA). We have determined the crystal structure of CyoA and purpleThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance w...

The genome of Azotobacter vinelandii contains DNA sequences homologous to the structural genes for the Escherichia coli cytochrome bd terminal oxidase complex. Two recombinant clones bearing cydA-and cydB-like sequence were isolated from an A. vinelandii gene library and subcloned into the plasmid vector pACYC184. Physical mapping demonstrated that the cydA-and cydB-like regions in A. vinelandii are contiguous. The cydAB and flanking DNA was mutagenized by the insertion of TnS-B20. Mutations in the cydB-hybridizing region resulted in the loss of spectral features associated with cytochromes b595 and d. A new locus, cydB, encoding cytochromes b595 and d in A. vinelandii is proposed. A second region adjacent to cydB was also involved in expression of the cytochrome bd complex in A. vinelandii, since mutations in this region resulted in an increase in the levels of both cytochrome b595 and cytochrome d. The regions involved in expression of the cytochrome bd complex and cydB are transcribed in the same direction. Mutants deficient in cytochromes b595 and d were unable to grow on N-deficient medium when incubated in air but could fix nitrogen when the environmental 02 concentration was reduced to 1.5% (vol/vol). It is proposed that the branch of the respiratory chain terminated by the cytochrome bd complex supports the high respiration rates required for the respiratory protection of nitrogenase.

The retinoblastoma binding protein KDM5A removes methyl marks from lysine 4 of histone H3 (H3K4). Misregulation of KDM5A contributes to the pathogenesis of lung and gastric cancers. In addition to its catalytic jumonji C domain, KDM5A contains three PHD reader domains, commonly recognized as chromatin recruitment modules. It is unknown whether any of these domains in KDM5A have functions beyond recruitment and whether they regulate the catalytic activity of the demethylase. Here using biochemical and nuclear magnetic resonance (NMR)-based structural studies, we show that the PHD1 preferentially recognizes unmethylated H3K4 histone tail, product of KDM5A-mediated demethylation of tri-methylated H3K4 (H3K4me3). Binding of unmodified H3 peptide to the PHD1 stimulates catalytic domain-mediated removal of methyl marks from H3K4me3 peptide and nucleosome substrates. This positive-feedback mechanism—enabled by the functional coupling between a reader and a catalytic domain in KDM5A—suggests a model for the spread of demethylation on chromatin.