VEGFs activate 3 receptor tyrosine kinases, VEGFR-1, VEGFR-2, and VEGFR-3, promoting angiogenic and lymphangiogenic signaling. The extracellular receptor domain (ECD) consists of 7 Ig-homology domains; domains 2 and 3 (D23) represent the ligandbinding domain, whereas the function of D4-7 is unclear. Ligand binding promotes receptor dimerization and instigates transmembrane signaling and receptor kinase activation. In the present study, isothermal titration calorimetry showed that the Gibbs free energy of VEGF-A, VEGF-C, or VEGF-E binding to D23 or the full-length ECD of VEGFR-2 is dominated by favorable entropic contribution with enthalpic penalty. The free energy of VEGF binding to the ECD is 1.0-1.7 kcal/mol less favorable than for binding to D23. A model of the VEGF-E/ VEGFR-2 ECD complex derived from smallangle scattering data provided evidence for homotypic interactions in D4-7. We also solved the crystal structures of complexes between VEGF-A or VEGF-E with D23, which revealed comparable binding surfaces and similar interactions between the ligands and the receptor, but showed variation in D23 twist angles. The energetically unfavorable homotypic interactions in D4-7 may be required for re-orientation of receptor monomers, and this mechanism might prevent ligand-independent activation of VEGFR-2 to evade the deleterious consequences for blood and lymph vessel homeostasis arising from inappropriate receptor activation. (Blood. 2012;119(7):1781-1788) IntroductionA plethora of growth factors, such as angiopoietins, VEGF family ligands, platelet-derived growth factors, fibroblast growth factors, and hepatocyte growth factors regulate blood and lymph vessel formation and homeostasis (reviewed in Cao 1 ). VEGFs represent a large family of ligands: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and PlGF, which bind to and activate in a combinatorial fashion 3 type V receptor tyrosine kinases (RTKs), VEGFR-1, VEGFR-2, and VEGFR-3, which give rise to highly specific signal output. In mammals, VEGF-A signaling through VEGFR-2 is the major angiogenic signaling pathway, but VEGF-C plays essential, and in some cases complementary, roles in the activation of this receptor (reviewed in Grünewald et al 2 ). The mechanism by which VEGFRs are activated is not understood in molecular detail, but clearly represents one of the many variations of RTK activation. In general, signaling by RTKs requires ligand-mediated dimerization with precise positioning of receptor subunits in active dimers. Dimeric ligand/receptor complexes subsequently initiate transmembrane signaling, resulting in the activation of the intracellular tyrosine kinase domains. 3,4 Active VEGFRs instigate cell signaling and promote endothelial cell migration and proliferation, as well as vessel fenestration and permeabilization. 5,6 The extracellular domain (ECD) of VEGFRs consists of 7 Ig-homology domains. The first 3 domains mediate ligand binding, 7,8 whereas the membrane proximal domains are involved in ligand-induced receptor dimerization. 7,9 Homotypic receptor i...

Hydrophobins are a group of surface-active fungal proteins known to adsorb to the air/water interface and self-assemble into highly crystalline films. We characterized the self-assembled protein films of two hydrophobins, HFBI and HFBII from Trichoderma reesei, directly at the air/water interface using Brewster angle microscopy, grazing-incidence X-ray diffraction, and reflectivity. Already in zero surface pressure, HFBI and HFBII self-assembled into micrometer-sized rafts containing hexagonally ordered two-dimensional crystallites with lattice constants of 55 A and 56 A, respectively. Increasing the pressure did not change the ordering of the proteins in the crystallites. According to the reflectivity measurements, the thicknesses of the hydrophobin films were 28 A (HFBI) and 24 A (HFBII) at 20 mN/m. The stable films could also be transferred to a silicon substrate. Modeling of the diffraction data indicated that both hydrophobin films contained six molecules in the unit cell, but the ordering of the molecules was somewhat different for HFBI and HFBII, suggesting specific protein-protein interactions.

The structural investigation of poly(9,9-bis(2-ethylhexyl)fluorene-2,7-diyl) (PF2/6) in aligned thin films is presented. Formation of a thickness dependent triaxial texturing is identified in thermotropically aligned films. X-ray reflectivity measurements reveal good macroscopic quality, and polarized photoluminescence and dichroic ratios in absorption indicate clear axial alignment. Grazing-incidence X-ray diffraction shows axially aligned mesomorphic structure with a distinct arrangement of helices and large correlation lengths, indicating a high local lateral order. Theoretical models produced using molecular mechanics methods suggest 5/2-helicity. The polymer chains are parallel to the substrate in the c direction. In particular, the hexagonal-like cells are flattened in the direction of the surface normal and reveal two kinds of coexistent crystallites, a multiple orientation where the greater proportion of the crystallites have one crystal axis a perpendicular to the substrate surface, whereas a smaller proportion is aligned with the crystal axis a parallel to the surface. In thinner films the former class of orientation is usually dominant, while the proportion of the parallel orientation type increases with prolonged annealing.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1-3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4-7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.signal transduction | receptor tyrosine kinase V EGFs stimulate angiogenesis and lymphangiogenesis via VEGF receptors (VEGFRs) in endothelial cells. VEGF-A signaling is mediated predominantly through activation of VEGFR-2, resulting in sprouting of blood vessels from preexisting vasculature (1). In contrast, VEGFR-1 seems to have an inhibitory role by sequestering VEGF-A and thereby preventing its interaction with VEGFR-2 (2). On the other hand, VEGFR-3 plays an indispensable role in lymphangiogenesis (3). VEGFRs are involved in various pathological conditions, including solid tumor growth, tumor metastasis, and vascular retinopathies (4, 5).VEGF-C and VEGF-D compose a VEGFR-3-specific subfamily of VEGFs. They are produced with large N-and C-terminal propeptides and gain activity toward VEGFR-3 and VEGFR-2 on proteolytic processing (reviewed in ref. 5). VEGFR-3 maturation involves proteolytic cleavage of the extracellular domain (ECD) in D5 (6-8). Both VEGF-C and VEGFR-3 also interact with the coreceptor neuropilin-2 (9). Loss of the Vegfc gene results in embryonic lethality owing to a lack of lymphatic vessel formation (10), whereas mutations that interfere with VEGFR-3 signaling have been associated with hereditary lymphedema, and mice deficient in Vegfr3 die in utero due to abnormal development of the blood vasculature (11, 12). VEGFR-3 and its heterodimers with VEGFR-2 are also important for sprouting angiogenesis and vascular network formation (13-15).VEGFRs are type V rece...

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases: VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single-particle electron microscopy, small-angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here, we describe the structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A at 4 Å resolution. We combined X-ray crystallography, single-particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in immunoglobulin homology domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts and identify them as potential therapeutic sites to allosterically inhibit VEGFR-1 activity.

Hydrophobins are a group of very surface-active, fungal proteins known to self-assemble on various hydrophobic/hydrophilic interfaces. The self-assembled films coat fungal structures and mediate their attachment to surfaces. Hydrophobins are also soluble in water. Here, the association of hydrophobins HFBI and HFBII from Trichoderma reesei in aqueous solution was studied using small-angle x-ray scattering. Both HFBI and HFBII exist mainly as tetramers in solution in the concentration range 0.5-10 mg/ml. The assemblies of HFBII dissociate more easily than those of HFBI, which can tolerate changes of pH from 3 to 9 and temperatures in the range 5 degrees C-60 degrees C. The self-association of HFBI and HFBII is mainly driven by the hydrophobic effect, and addition of salts along the Hofmeister series promotes the formation of larger assemblies, whereas ethanol breaks the tetramers into monomers. The possibility that the oligomers in solution form the building blocks of the self-assembled film at the air/water interface is discussed.

Receptor tyrosine kinases play essential roles in tissue development and homeostasis, and aberrant signaling by these molecules is the basis of many diseases. Understanding the activation mechanism of these receptors is thus of high clinical relevance. We investigated vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), which regulate blood and lymph vessel formation. We analyzed the structural changes in the extracellular receptor domain that were induced by ligand binding and that represent the initial step in transmembrane signaling, culminating in the activation of the intracellular receptor kinase domain. High-resolution structural information for the ligand binding domain became available recently, but the flexibility of the extracellular domain and inhomogeneous glycosylation of VEGFRs have prevented the production of highly diffracting crystals of the entire extracellular domain so far. Therefore, we chose to further investigate VEGFR structure by small-angle X-ray scattering in solution (SAXS). SAXS data were combined with independent distance restraint determination obtained by mass spectrometric analysis of chemically cross-linked ligand/receptor complexes. With these data, we constructed a structural model of the entire extracellular receptor domain in the unbound form and in complex with VEGF.

Hydrophobins are small fungal surface active proteins that self-assemble at interfaces into films with nanoscale structures. The hydrophobin HFBI from Trichoderma reesei has been shown to associate in solution into tetramers but the role of this association on the function of HFBI has remained unclear. We produced two HFBI variants that showed a significant shift in solution association equilibrium towards the tetramer state. However, this enhanced solution association did not alter the surface properties of the variant HFBIs. The results show that there is not a strong relationship between HFBI solution association state and surface properties such as surface activity.