Co-crystal structures of P-glycoprotein with a series of engineered ligands reveal multiple ligand-binding modes, a ligand-binding site on the outer surface of the transporter and a conformational change that may couple to ATP hydrolysis.

Common seafood pollutants inhibit a crucial cellular defense protein.

Humans have three functioning genes that code for copper-containing amine oxidases. The product of the AOC1 gene is a so-called diamine oxidase (hDAO), named for its substrate preference for diamines, particularly histamine. hDAO has been cloned and expressed in insect cells and the structure of the native enzyme determined by X-ray crystallography to a resolution of 1.8 Å. The homodimeric structure has the archetypal amine oxidase fold. Two active sites, one in each subunit, are characterized by the presence of a copper ion and a topaquinone residue formed by the post-translational modification of a tyrosine. Although hDAO shares 37.9 % sequence identity with another human copper amine oxidase, semicarbazide sensitive amine oxidase or vascular adhesion protein-1, its substrate binding pocket and entry channel are distinctly different in accord with the different substrate specificities. The structures of two inhibitor complexes of hDAO, berenil and pentamidine, have been refined to resolutions of 2.1 Å and 2.2 Å, respectively. They bind non-covalently in the active site channel. The inhibitor binding suggests that an aspartic acid residue, conserved in all diamine oxidases but absent from other amine oxidases, is responsible for the diamine specificity by interacting with the second amino group of preferred diamine substrates.

The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA (Nx)AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5 splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine ''ladder.'' A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.protein structure ͉ RanBP2 zinc fingers ͉ RNA-binding proteins ͉ splicing A lmost all human genes are thought to be alternatively spliced, and it has been estimated that at least 15% of human diseases are associated with changes in RNA processing (1). The selection of splice sites is influenced heavily by the binding of accessory splicing factors to regulatory sequences in the pre-mRNA. SR proteins are splicing factors that contain a C-terminal Arg/Ser-rich (RS) domain and either 1 or 2 N-terminal RNA recognition motifs (RRMs) (2). They play crucial roles in constitutive and alternative splicing, promoting recognition of splice sites by binding to exonic splicing enhancers (ESEs). RRM domains bind ssRNA with high affinity in a sequence-specific manner, whereas RS domains appear to facilitate both protein-protein and protein-RNA interactions (3, 4). Other RS domain-containing proteins that lack a canonical RRM, termed ''SR-like'' proteins (see, for example, ref. 5) are also known to play roles in splicing.ZRANB2 (Zis, ZNF265) is an SR-like nuclear protein that is expressed in most tissues and is conserved between nematodes and mammals. It interacts with the spliceosomal proteins U1-70K and U2AF 35 and can alter the distribution of splice variants of GluR-B, SMN2, and Tra2␤ in minigene reporter assays (6, 7). As such, ZRANB2 appears to regulate splice site choice. However, in place of the canonical RNA-binding RRM domains, ZRANB2 displays 2 N-terminal RanBP2-type zinc fingers (ZnFs).RanBP2-type ZnF domains are defined by the consensus sequence W-X-C-X 2-4 -C-X 3 -N-X 6 -C-X 2 -C. They occur multiple times in at least 21 human proteins, and the fold comprises 2 distorted ␤-hairpins sandwiching a central tryptophan residue and ...