Members of the Wiskott-Aldrich Syndrome Protein (WASP) family control cytoskeletal dynamics by promoting actin filament nucleation by the Arp2/3 complex. The WASP relative, WAVE, regulates lamellipodia formation within a 400 kDa, hetero-pentameric WAVE Regulatory Complex (WRC). The WRC is inactive toward the Arp2/3 complex, but can be stimulated by the Rac GTPase, kinases and phosphatidylinositols. We report the 2.3 Å crystal structure of the WRC and complementary mechanistic analyses. The structure shows that the activity-bearing VCA motif of WAVE is sequestered by a combination of intramolecular and intermolecular contacts within the WRC. Rac and kinases appear to destabilize a WRC element that is necessary for VCA sequestration, suggesting how these signals stimulate WRC activity toward the Arp2/3 complex. Spatial proximity of the Rac binding site and a large basic surface of the WRC suggests how the GTPase and phospholipids could cooperatively recruit the complex to membranes.

A novel and general approach to scaling diffraction intensities is presented. The method minimizes the disagreement among multiple measurements of symmetry-related re¯ections using a stable re®nement procedure. The scale factors are described by a¯exible exponential function that allows different scaling corrections to be chosen and combined according to the needs of the experiment. The scaling model presented here includes: scale and temperature factor per batch of data; temperature factor as a continuous function of the radiation dose; absorption in the crystal; uneven exposure within a single diffraction image; and corrections for phenomena that depend on the diffraction peak position on the detector. This scaling model can be extended to include additional corrections for various instrumental and data-collection problems.

Summary The VP35 protein encoded by the highly pathogenic Ebola virus facilitates immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I like receptors. Here we report the crystal structure of Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which reveals how VP35-dsRNA interactions contribute to immune evasion, and corresponding in vivo studies. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are “end-capped” by a pocket of hydrophobic residues that mimics blunt end dsRNA recognition by RIG-I-like receptors. Residues that are critical for RNA binding are also important for interferon inhibition in vivo, but not for viral polymerase co-factor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.

Histone methylation regulates diverse chromatin-templated processes, including transcription. Many transcriptional corepressor complexes contain lysine-specific demethylase 1 (LSD1) and CoREST that collaborate to demethylate mono- and dimethylated H3-K4 of nucleosomes. Here, we report the crystal structure of the LSD1-CoREST complex. LSD1-CoREST forms an elongated structure with a long stalk connecting the catalytic domain of LSD1 and the CoREST SANT2 domain. LSD1 recognizes a large segment of the H3 tail through a deep, negatively charged pocket at the active site and possibly a shallow groove on its surface. CoREST SANT2 interacts with DNA. Disruption of the SANT2-DNA interaction diminishes CoREST-dependent demethylation of nucleosomes by LSD1. The shape and dimension of LSD1-CoREST suggest its bivalent binding to nucleosomes, allowing efficient H3-K4 demethylation. This spatially separated, multivalent nucleosome binding mode may apply to other chromatin-modifying enzymes that generally contain multiple nucleosome binding modules.

ATP binding cassette (ABC) transporters play critical roles in maintaining sterol balance in higher eukaryotes. The ABCG5/ABCG8 heterodimer (G5G8) mediates excretion of neutral sterols in liver and intestines1–5. Mutations disrupting G5G8 cause sitosterolaemia, a disorder characterized by sterol accumulation and premature atherosclerosis. Here we use crystallization in lipid bilayers to determine the X-ray structure of human G5G8 in a nucleotide-free state at 3.9 Å resolution, generating the first atomic model of an ABC sterol transporter. The structure reveals a new transmembrane fold that is present in a large and functionally diverse superfamily of ABC transporters. The transmembrane domains are coupled to the nucleotide-binding sites by networks of interactions that differ between the active and inactive ATPases, reflecting the catalytic asymmetry of the transporter. The G5G8 structure provides a mechanistic framework for understanding sterol transport and the disruptive effects of mutations causing sitosterolaemia.