SUMMARY Tetraspanins comprise a diverse family of four-pass transmembrane proteins that play critical roles in the immune, reproductive, genitourinary, and auditory systems. Despite their pervasive roles in human physiology, little is known about the structure of tetraspanins or the molecular mechanisms underlying their various functions. Here we report the crystal structure of a full-length tetraspanin, human CD81. The transmembrane segments of CD81 pack as two largely separated pairs of helices, capped by the large extracellular loop (EC2) at the outer membrane leaflet. The two pairs of helices converge at the inner leaflet to create an intramembrane pocket with additional electron density corresponding to a bound cholesterol molecule within the cavity. Molecular dynamics simulations identify an additional conformation in which EC2 separates substantially from the transmembrane domain. Cholesterol binding appears to modulate CD81 activity in cells, suggesting a potential mechanism for regulation of tetraspanin function.

SUMMARY The endothelial specific receptor tyrosine kinase Tie2, and the orphan receptor tyrosine kinase, Tie1, are essential for endothelial cell proliferation, migration, and survival during angiogenesis. Despite their considerable similarity, experiments with Tie1 or Tie2 deficient mice highlight distinct functions for these two receptors in vivo. However, Tie1 cooperates with Tie2 during Angiopoietin signaling, demonstrating a degree of functional overlap between the two receptor-systems. Tie2 is further unique among receptor tyrosine kinases with respect to its structurally homologous ligands. Angiopoietin-2 and -3 can function as agonists or antagonists depending upon the local environment, while Angiopoietin-1 and -4, are constitutive agonists. To address the role of Tie1 in Angiopoietin-mediated Tie2 signaling, and determine the basis for the unique behavior of the individual Angiopoietins, we used an in vivo FRET-based proximity assay to monitor Tie1 and Tie2 localization and association in the presence and absence of ligands. Here, we provide direct evidence for Tie1-Tie2 complex formation on the endothelial cell surface and identify molecular surface areas essential for receptor-receptor recognition. We further demonstrate that the Tie1-Tie2 interactions are dynamic, inhibitory, and differentially modulated by Angiopoietin-1 and -2, thereby providing a discrete molecular mechanism for the observed variations in Angiopoietin function. Based upon the available data, we propose a unified model for Angiopoietin-induced Tie2 signaling which highlights the essential role of Tie1 in vascular homeostasis.

SUMMARY Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer’s precursor protein, APP. We present here the X-ray crystal structure of the ADAM10 ectodomain, which together with biochemical and cellular studies reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture, in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.

Angiogenesis is a complex cellular process involving multiple regulatory growth factors and growth factor receptors. Among them, the ligands for the endothelial-specific tunica intima endothelial receptor tyrosine kinase 2 (Tie2) receptor kinase, angiopoietin-1 (Ang1) and Ang2, play essential roles in balancing vessel stability and regression during both developmental and tumorinduced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cellsignaling characteristics. Here, we present the crystal structures of Ang1 both unbound and in complex with the Tie2 ectodomain.Comparison of the Ang1-containing structures with their Ang2-containing counterparts provide insight into the mechanism of receptor activation and reveal molecular surfaces important for interactions with Tie2 coreceptors and associated signaling proteins. Using structure-based mutagenesis, we identify a loop within the angiopoietin P domain, adjacent to the receptor-binding interface, which confers the specific agonist/antagonist properties of the molecule. We demonstrate using cell-based assays that an Ang2 chimera containing the Ang1 loop sequence behaves functionally similarly to Ang1 as a constitutive Tie2 agonist, able to efficiently dissociate the inhibitory Tie1/Tie2 complex and elicit Tie2 clustering and downstream signaling. cellular signaling | Tie receptor tyrosine kinase | X-ray crystallography P roper development of the cardiovascular system involves two highly integrated and dynamic processes: vasculogenesis and angiogenesis (1). Vasculogenesis involves the in situ differentiation of endothelial cells from precursor angioblasts and results in their subsequent migration and proliferation, leading to the formation of the endocardium of the heart, as well as the major primitive blood vessels (2-6). Angiogenesis occurs following vasculogenesis and results in the remodeling and extension of the endothelial tubes into the adult microvasculature, as well as in the growth and expansion of vessels into organs including the kidney and brain. Endothelial cells lining these newly formed vessels are surrounded by support cells, such as smooth muscle cells and pericytes, which serve to regulate blood flow and provide survival signals (7). Angiogenesis predominantly takes place during embryonic development, although it also recurs during wound repair in adulthood. Its role is essential for tumor development, as well as metastasis and, therefore, represents a viable target for therapeutic intervention (8).The angiopoietins are a small set of growth factor ligands for the tunica intima endothelial receptor tyrosine kinase 2 (Tie2) endothelial-specific receptor tyrosine kinase and are critical for both development and pathological angiogenesis (9-11). The prototypic family member, angiopoietin-1 (Ang1), is a Tie2 agonist, whereas the highly homologous Ang2 is a context-dependent agonist/antagonist. All angiopoietin family members, Ang1 to -4, contain an amino-terminal "superclustering" and...

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria in order to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSH. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. Strikingly, this inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.