ABSTRACT
Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (VEGFRs). The extracellular domain of VEGFRs consists of seven Ig-homology domains, of which D2-3 form the ligand-binding site, while the membrane proximal domains D4-7 are involved in homotypic interactions in ligand-bound receptor dimers. Based on low-resolution structures, we identified allosteric sites in D4-5 and D7 of vascular endothelial growth factor receptor 2 (VEGFR-2) accomplishing regulatory functions. Allosteric inhibition of VEGFR-2 signaling represents an attractive option for the treatment of neovascular diseases. We showed earlier that DARPin® binders to domains D4 or D7 are potent VEGFR-2 inhibitors. Here we investigated in detail the allosteric inhibition mechanism of the domain D4 binding inhibitor D4b. The 2.38 Å crystal structure of D4b in complex with VEGFR-2 D4-5, the first high-resolution structure of this VEGFR-2 segment, indicates steric hindrance by D4b as the mechanism of inhibition of receptor activation. At the cellular level, D4b triggered quantitative internalization of VEGFR-2 in the absence of ligand and thus clearance of VEGFR-2 from the surface of endothelial cells. The allosteric VEGFR-2 inhibition was sufficiently strong to efficiently inhibit the growth of human endothelial cells at suboptimal dose in a mouse xenograft model in vivo, underlining the therapeutic potential of the approach.
Subject(s)
Angiogenesis Inhibitors , Drug Delivery Systems , Human Umbilical Vein Endothelial Cells , Neovascularization, Pathologic , Signal Transduction , Vascular Endothelial Growth Factor A , Allosteric Regulation/drug effects , Allosteric Site , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacology , Animals , Crystallography, X-Ray , HEK293 Cells , Heterografts , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/pathology , Human Umbilical Vein Endothelial Cells/transplantation , Humans , Mice , Mice, SCID , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Protein Binding , Signal Transduction/drug effects , Signal Transduction/genetics , Swine , Transplantation, Heterologous , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/chemistry , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/chemistry , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.
Subject(s)
Algal Proteins/metabolism , Centrioles/metabolism , Chlamydomonas reinhardtii/metabolism , Microtubules/metabolism , Algal Proteins/chemistry , Algal Proteins/genetics , Blotting, Western , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Centrioles/chemistry , Centrioles/ultrastructure , Chlamydomonas reinhardtii/genetics , Crystallography, X-Ray , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Microscopy, Atomic Force , Microscopy, Electron , Microscopy, Fluorescence , Microtubules/chemistry , Microtubules/ultrastructure , Models, Molecular , Molecular Conformation , Mutation , Protein Multimerization , Protein Structure, Tertiary , RNA InterferenceABSTRACT
Botulinum neurotoxin A causes botulism but is also used for medical and cosmetic applications. A detailed molecular understanding of BoNT/A--host receptor interactions is therefore fundamental for improving current clinical applications and for developing new medical strategies targeting human disorders. Towards this end, we recently solved an X-ray crystal structure of BoNT/A1 in complex with its neuronal protein receptor SV2C. Based on our findings, we discuss the potential implications for BoNT/A function.