The biological effects of the ISG15 protein arise in part from its conjugation to cellular targets as a primary response to interferon-alpha/beta induction and other markers of viral or parasitic infection. Recombinant full-length ISG15 has been produced for the first time in high yield by mutating Cys78 to stabilize the protein and by cloning in a C-terminal arginine cap to protect the C terminus against proteolytic inactivation. The cap is subsequently removed with carboxypeptidase B to yield mature biologically active ISG15 capable of stoichiometric ATP-dependent thiolester formation with its human UbE1L activating enzyme. The three-dimensional structure of recombinant ISG15C78S was determined at 2.4-A resolution. The ISG15 structure comprises two beta-grasp folds having main chain root mean square deviation (r.m.s.d.) values from ubiquitin of 1.7 A (N-terminal) and 1.0 A (C-terminal). The beta-grasp domains pack across two conserved 3(10) helices to bury 627 A2 that accounts for 7% of the total solvent-accessible surface area. The distribution of ISG15 surface charge forms a ridge of negative charge extending nearly the full-length of the molecule. Additionally, the N-terminal domain contains an apolar region comprising almost half its solvent accessible surface. The C-terminal domain of ISG15 was superimposed on the structure of Nedd8 (r.m.s.d. = 0.84 A) bound to its AppBp1-Uba3 activating enzyme to model ISG15 binding to UbE1L. The docking model predicts several key side-chain interactions that presumably define the specificity between the ubiquitin and ISG15 ligation pathways to maintain functional integrity of their signaling.

Background:The mechanism of E6AP has been previously defined by the structure of a substrate-bound intermediate. Results:Rate studies of polyubiquitin chain formation preclude the canonical model based on the crystal structure. Conclusion: Kinetics define a mechanism requiring two functionally distinct E2ϳubiquitin thioester binding sites. Significance: A general mechanism for Hect ligase polyubiquitin chain formation is defined for E6AP based on empirical rate measurements.

Here we introduce a database of calibrated natural images publicly available through an easy-to-use web interface. Using a Nikon D70 digital SLR camera, we acquired about six-megapixel images of Okavango Delta of Botswana, a tropical savanna habitat similar to where the human eye is thought to have evolved. Some sequences of images were captured unsystematically while following a baboon troop, while others were designed to vary a single parameter such as aperture, object distance, time of day or position on the horizon. Images are available in the raw RGB format and in grayscale. Images are also available in units relevant to the physiology of human cone photoreceptors, where pixel values represent the expected number of photoisomerizations per second for cones sensitive to long (L), medium (M) and short (S) wavelengths. This database is distributed under a Creative Commons Attribution-Noncommercial Unported license to facilitate research in computer vision, psychophysics of perception, and visual neuroscience.

Background: E6AP/UBE3A is a Hect ligase implicated in neurodevelopment and cell transformation. Results: Kinetic/biophysical analyses demonstrate that E6AP oligomerization is required for polyubiquitin degradation signal assembly and that changes in oligomerization regulates such activity. Conclusion: E6AP oligomerization accounts for opposing effects of mutation and HPV16/18 E6 protein.Significance: These findings explain in part the etiology of specific Angelman syndrome mutations and E6-mediated cell transformation.

Background: Ubiquitin carrier protein (E2) recognition by ubiquitin activating enzyme (E1) defines fidelity in subsequent conjugation reactions. Results: E2 transthiolation kinetics identify structural features defining the specificity of E1-E2 binding. Conclusion: E2 paralogs contain a conserved E1 binding motif, and the E1 ␤-grasp domain is a specificity filter for E2 binding. Significance: This defines structural features determining ubiquitin conjugation fidelity.