Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP-and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (⌬F508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of ⌬F508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the ⌬F508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of ⌬F508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the ⌬F508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.Cystic fibrosis causes lung, liver, pancreas, and reproductive tract disorders, typically leading to death prior to middle age from deterioration in pulmonary function (1). CFTR 1 protein is composed of two membrane spanning domains (MSD1 and MSD2), two nucleotide-binding domains (NBD1 and NBD2), and a regulatory region (R). Although it functions as an ATPgated anion channel, CFTR is a member of the ATP-binding cassette (ABC) transporter superfamily (2) based on high sequence similarity between the NBDs and canonical ABC domains. Understanding the exact molecular pathology caused by the ⌬F508 mutation in CFTR is of great importance in the development of drugs to treat cystic fibrosis because of the prevalence of this mutation in the human population. ⌬F508 CFTR fails to mature appropriately in the endoplasmic reticulum and is poorly populated in the epithelial membrane (3-6). It has been proposed that the primary effect of the ⌬F508 mutation is to cause misfolding of NBD1, which leads to aberrant transport and ultimately targeted proteolytic degradation of CFTR (7,8). Channels harboring the deletion show enhanced sensitivity to proteolytic degradation (9) but have at least partial wild-type chloride conductance properties (4, 10). Canonical ABC domain structures are composed of three subdomains, a central F1-type ATP-binding core subdomain, an antiparallel ␤-sheet (ABC␤) s...

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.

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SignificanceIndoleamine 2,3-dioxygenase (IDO1) is a heme protein that catalyzes the dioxygenation of tryptophan. Cells expressing this activity are able to profoundly alter their surrounding environment to suppress the immune response. Cancer cells exploit this pathway to avoid immune-mediated destruction. Through a range of kinetic, structural, and cellular assays, we show that two classes of highly selective inhibitors of IDO1 act by competing with heme binding to apo-IDO1. This shows that IDO1 is dynamically bound to its heme cofactor in what is likely a critical step in the regulation of this enzyme. These results have elucidated a previously undiscovered role for the ubiquitous heme cofactor in immune regulation, and it suggests that other heme proteins in biology may be similarly regulated.

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higherorder NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.antiinfluenza | oligomerization | polymerase inhibitor | protein-protein interaction | cooperative inhibition

The crystal structures of both Nova KH3 domains are similar to the previously determined NMR structures. The most significant differences among the KH domains involve changes in the positioning of one or more of the alpha helices with respect to the betasheet, particularly in the NMR structure of the KH1 domain of the Fragile X disease protein FMR-1. Loop regions in the KH domains are clearly visible in the crystal structure, unlike the NMR structures, revealing the conformation of the invariant Gly-X-X-Gly segment that is thought to participate in RNA-binding and of the variable region. The tetrameric arrangements of the Nova KH3 domains provide insights into how KH domains may interact with each other in proteins containing multiple KH motifs.

The Nova family of proteins are target antigens in the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia and contain K-homology (KH)-type RNA binding domains. The Nova-1 protein has recently been shown to regulate alternative splicing of the ␣2 glycine receptor subunit pre-mRNA by binding to an intronic element containing repeats of the tetranucleotide UCAU. Here, we have used selection-amplification to demonstrate that the KH3 domain of Nova recognizes a single UCAY element in the context of a 20-base hairpin RNA; the UCAY tetranucleotide is optimally presented as a loop element of the hairpin scaffold and requires protein residues C-terminal to the previously defined KH domain. These results suggest that KH domains in general recognize tetranucleotide motifs and that biological RNA targets of KH domains may use either RNA secondary structure or repeated sequence elements to achieve high affinity and specificity of protein binding. In eukaryotes, transcription of pre-mRNA is followed by a series of processing steps, including mRNA splicing, polyadenylation, export, localization, translation, and degradation. RNA-protein interactions are central in all of these pathways, and RNA binding proteins are often key regulators of posttranscriptional gene expression. The elucidation of how RNA binding proteins interact with RNA is thus critical to the understanding of these aspects of mRNA processing.One of the most common protein motifs involved in RNA binding is the K-homology (KH) domain, originally described in the protein hnRNP-K (1). The KH domain consists of approximately 70 amino acids and includes a conserved hydrophobic core, an invariant Gly-X-X-Gly motif, and an additional variable segment; isolated KH domains of approximately this length are competent to bind ribohomopolymers (2-4). Over 50 KH domain-containing proteins have been identified; notably, many of these proteins contain multiple KH domains and͞or other known RNA binding domains, such as the RNA recognition motif. NMR structural studies of individual KH domains revealed a conserved ␤␣␣␤␤␣ fold (3, 5), and high-resolution x-ray structures have additionally permitted the visualization of the Gly-X-X-Gly and variable loops (6).How KH domain proteins function requires understanding how they interact with RNA and the roles these interactions play in the context of a presumably complex and multicomponent regulatory system. The mammalian KH domain proteins KSRP and SF1, the yeast KH protein MER-1, and the Drosophila KH domain protein PSI have all been implicated in regulating mRNA splicing (7-10). The heterogeneous nuclear RNP proteins hnRNP K and hnRNP E have been shown to play important roles in mRNA stabilization and mRNA translational control (11-13). The proteins ZBP-1 and Vera, which each contain two RNA recognition motif domains and four KH domains, have been shown to play a role in localizing specific mRNAs within the cell cytoplasm (14, 15). The absence of the mammalian KH domain protein FMR-1 is associated with the fragile-x mental...