SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 A apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold.

Engineered pores have several advantages as potential sensor elements: sensitivity is in the nanomolar range; analyte binding is rapid (diffusion limited in some cases) and reversible; strictly selective binding is not required because single-channel recordings are rich in information; and for a particular analyte, the dissociation rate constant, the extent of channel block and the voltage-dependence of these parameters are distinguishing, while the frequency of partial channel block reflects the analyte concentration. A single sensor element might, therefore, be used to quantitate more than one analyte at once. The approach described here can be generalized for additional analytes.

No abstract

No abstract

Elucidation of the accurate subunit stoichiometry of oligomeric membrane proteins is fraught with complexities. The interpretations of chemical cross-linking, analytical ultracentrifugation, gel filtration, and low-resolution electron microscopy studies are often ambiguous. Staphylococcal a-hemolysin (crHL), a homooligomeric toxin that forms channels in cell membranes, was believed to possess six subunits arranged around a sixfold axis of symmetry. Here, we report that analysis of x-ray diffraction data and chemical modification experiments indicate that the aHlL oligomer is a heptamer. Self-rotation functions calculated using x-ray diffraction data from single crystals of acHL oligomers show a sevenfold axis of rotational symmetry. The aHiL pore formed on rabbit erythrocyte membranes was determined to be a heptamer by electrophoretic separation of aHL heteromers formed from subunits with the charge of wild-type aHL and subunits with additional negative charge generated by targeted chemical modification of a single-cysteine mutant. These data establish the heptameric oligomerization state of the aHlL transmembrane pore both in three-dimensional crystals and on a biological membrane.As exemplified by studies on cholera toxin (1), chemical cross-linking and electron microscopy may not provide precise and unequivocal information on the subunit stoichiometry of oligomeric proteins. Complete cross-linking of an oligomeric protein can give both cyclic and linear species, which may migrate differently on denaturing polyacrylamide gels, giving the false indication of an additional subunit (1). Dynamic or static rotational disorder about the axis of molecular symmetry in two-dimensional crystals can yield misleading diffraction data (1). Resolution of the subunit composition of cholera toxin was achieved by the collection and analysis of high-resolution x-ray diffraction data (2). A second example is aerolysin, a pore-forming protein secreted by Aeromonas hydrophila. Aerolysin was thought to form a pentamer or a hexamer (3) until a recent examination by rotational correlation analysis of particles in averaged electron micrographs suggested that it is a heptamer (4). However, this conclusion is being reevaluated (5). The gap junction connexon provides another example of an oligomeric membrane protein for which the number of subunits is still unclear. The connexon was believed to be a hexamer (6), but recent work suggests that it may be a pentamer (7).Reservations concerning the reliability of methods employed in determining the subunit stoichiometry of other membrane proteins have led us to reexamine the quaternary structure of a-hemolysin (aHL), a protein that is a model system for studying membrane protein assembly (8-11), and that has potential applications in biotechnology [e.g., as a component of immunotoxins (12) or an element in biosensors (13)]. The aHL polypeptide of 293 amino acids is secreted by Staphylococcus aureus as a water-soluble monomer and assembles upon contact with lipid bilayers or the deterg...

Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.

No abstract

The conformations of loops are determined by the water-mediated interactions between amino acid residues. Energy functions that describe the interactions can be derived either from physical principles (physicalbased energy function) or statistical analysis of known protein structures (knowledge-based statistical potentials). It is commonly believed that statistical potentials are appropriate for coarse-grained representation of proteins but are not as accurate as physical-based potentials when atomic resolution is required. Several recent applications of physical-based energy functions to loop selections appear to support this view. In this article, we apply a recently developed DFIRE-based statistical potential to three different loop decoy sets (RAPPER, Jacobson, and Forrest-Woolf sets). Together with a rotamer library for side-chain optimization, the performance of DFIRE-based potential in the RAPPER decoy set (385 loop targets) is comparable to that of AMBER/GBSA for short loops (two to eight residues). The DFIRE is more accurate for longer loops (9 to 12 residues). Similar trend is observed when comparing DFIRE with another physicalbased OPLS/SGB-NP energy function in the large Jacobson decoy set (788 loop targets). In the ForrestWoolf decoy set for the loops of membrane proteins, the DFIRE potential performs substantially better than the combination of the CHARMM force field with several solvation models. The results suggest that a single-term DFIRE-statistical energy function can provide an accurate loop prediction at a fraction of computing cost required for more complicate physical-based energy functions. A Web server for academic users is established for loop selection at the softwares/services section of the Web site http://theory.med. buffalo.edu/.