Transient interactions of platelet-receptor glycoprotein Ibalpha (GpIbalpha) and the plasma protein von Willebrand factor (VWF) reduce platelet velocity at sites of vascular damage and play a role in haemostasis and thrombosis. Here we present structures of the GpIbalpha amino-terminal domain and its complex with the VWF domain A1. In the complex, GpIbalpha wraps around one side of A1, providing two contact areas bridged by an area of solvated charge interaction. The structures explain the effects of gain-of-function mutations related to bleeding disorders and provide a model for shear-induced activation. These detailed insights into the initial interactions in platelet adhesion are relevant to the development of antithrombotic drugs.

Slits are large multidomain leucine-rich repeat (LRR)-containing proteins that provide crucial guidance cues in neuronal and vascular development. More recently, Slits have been implicated in heart morphogenesis, angiogenesis, and tumor metastasis. Slits are ligands for the Robo (Roundabout) receptors, which belong to the Ig superfamily of transmembrane signaling molecules. The Slit-Robo interaction is mediated by the second LRR domain of Slit and the two N-terminal Ig domains of Robo, but the molecular details of this interaction and how it induces signaling remain unclear. Here we describe the crystal structures of the second LRR domain of human Slit2 (Slit2 D2), the first two Ig domains of its receptor Robo1 (Ig1-2), and the minimal complex between these proteins (Slit2 D2-Robo1 Ig1). Slit2 D2 binds with its concave surface to the side of Ig1 with electrostatic and hydrophobic contact regions mediated by residues that are conserved in other family members. Surface plasmon resonance experiments and a mutational analysis of the interface confirm that Ig1 is the primary domain for binding Slit2. These structures provide molecular insight into Slit-Robo complex formation and will be important for the development of novel cancer therapeutics.guidance cues ͉ neurons ͉ signaling B ilaterally symmetric nervous systems, such as those of insects and vertebrates, possess a special midline structure that establishes a partition between the left and right mirror-image halves. To connect and coordinate both sides, a subset of so-called commissural axons has to cross the midline. Developing commissural axons navigate through the embryo by processing and responding to a number of different signals in their immediate environment. Both Slit and Netrin and their receptors, Roundabout (Robo) and Deleted in Colorectal Carcinoma (DCC), provide key ligand-receptor interactions for this process during neuronal development, especially at the midline of the central nervous system of vertebrates and invertebrates (1, 2). The Slit-Robo signaling complex is also central to the development of blood vessels (3, 4) and some organs, for example, the heart (5, 6). In addition, Slit2 has been implicated in breast cancer metastasis (7) and Robo1 in heptacellular carcinoma (8).Three Slit proteins (Slit1-3) (9) and four Robo proteins (Robo1, Robo2, Robo3/Rig-1, and the vascular-specific Robo4/ magic Roundabout) (10-12) have been identified in mammals. Netrin and Slit1-3 are secreted by the midline cells, whereas DCC and Robo1-3 are expressed on the surface of growing axons (2). During neuronal development, the commissural axons are initially attracted to the midline through a Netrin-DCCmediated interaction (2). This attractive signal is subsequently silenced near the midline to allow crossing through a Slitmediated interaction between DCC and Robo (13). Slit-Robo signaling then induces repulsion, expelling the axons from the midline and preventing recrossing (1). In vertebrates, Robo3 is also proposed to have a role in midline crossing by antagon...

Factor B is the central protease of the complement system of immune defense. Here, we present the crystal structure of human factor B at 2.3-Å resolution, which reveals how the five-domain proenzyme is kept securely inactive. The canonical activation helix of the Von Willebrand factor A (VWA) domain is displaced by a helix from the preceding domain linker. The two helices conformationally link the scissile-activation peptide and the metal ion-dependent adhesion site required for binding of the ligand C3b. The data suggest that C3b binding displaces the three N-terminal control domains and reshuffles the two central helices.Reshuffling of the helices releases the scissile bond for final proteolytic activation and generates a new interface between the VWA domain and the serine protease domain. This allosteric mechanism is crucial for tight regulation of the complementamplification step in the immune response.Factor B is a tightly regulated, highly specific serine protease. In its activated form, it catalyzes the central amplification step of complement activation to initiate inflammatory responses, cell lysis, phagocytosis and B-cell stimulation 1,2 . Factor B is activated through an assembly process: it binds surface-bound C3b, or its fluid-phase counterpart C3(H 2 O), after which it is cleaved by factor D into fragments Ba (residues 1-234) and Bb (residues 235-739) 3,4 . Fragment Ba dissociates from the complex, leaving behind the alternative pathway C3 convertase complex C3b-Bb, which cleaves C3 into C3a and C3b (see Fig. 1a). This protease complex is intrinsically instable. Once dissociated from the complex, Bb cannot reassociate with C3b 5 . A similar C3 convertase complex is formed upon activation of the classical (antibody-mediated) and lectin-binding pathways, comprised of C4 and C2, which are homologous to C3 and factor B, respectively. The proenzyme factor B consists of three N-terminal complement control protein (CCP) domains, connected by a 45-residue linker to a VWA domain and a C-terminal serine protease (SP) domain, which carries the catalytic center (Fig. 1a). The VWA and SP domains form fragment Bb, and CCP1 through CCP3 and the linker form fragment Ba. Binding of factor B to C3b depends on elements in fragment Ba 6 and the Mg 2+ -dependent metal ion-dependent adhesion site (MIDAS) motif in the VWA domain of fragment Bb 7 . The VWA domain is structurally homologous to inserted (I) domains in integrins. In I domains, ligand binding to the MIDAS is coupled to a B10-Å shift of the a7 activation helix, with concomitant domain rearrangements that activate the integrins 8,9 . Structures of a truncated Bb fragment 10 and its full-length homolog C2a 11 show variable positions of the a7 activation helix affecting the orientation of the VWA and SP domains, which indicates that a related mechanism may occur in convertase formation and dissociation. These structures, however, do not reveal the regulation of the proteolytic activity of factor B. In particular, it is unclear how factor B is maintained in its in...

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombincleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors. (Blood. 2008;112: 2803-2809) Introduction TAFI 1,2 is a pro-metallocarboxypeptidase that links the coagulation and fibrinolytic systems. TAFI is activated by thrombin, the thrombin-thrombomodulin complex or plasmin. 3 Activated TAFI (TAFIa) inhibits plasmin-mediated blood clot lysis by removing C-terminal lysine residues from partially degraded fibrin that are required for positive feedback in tissue plasminogen-activator dependent plasmin generation. In addition, TAFIa has been implicated in modulation of the inflammatory response by inactivating bradykinin and the anaphylatoxins C3a and C5a. 4,5 Although it is a powerful antifibrinolytic agent, there are no known physiologic inhibitors of TAFIa. Instead, the half-life of TAFIa activity is regulated by its intrinsic instability. The inactivation rate, 5 to 10 minutes at 37°C, is highly temperature-dependent, suggesting that inactivation involves a large conformational change. 6 This is also suggested by the susceptibility of the inactive enzyme, TAFIai to proteolytic cleavage by thrombin at Arg302, a site that is cryptic in TAFI and TAFIa. 6,7 The stability of TAFIa is an important determinant for its antifibrinolytic potential because TAFIa inhibits fibrinolysis through a threshold-dependent mechanism. [8][9][10] Full-length TAFI consists of 401 amino acids divided into 2 domains: the first 92 amino acids form the activation peptide; the next 309 amino acids form the catalytic domain. The activation peptide restricts substrate access to the catalytic cleft in the zymogen. TAFI is activated through cleavage at Arg92, which releases the activation peptide.TAFI is highly homologous to the pancreatic procarboxypeptidases with 42% sequence identity to human procarboxypeptidase B (pro...