Structural analysis of vascular endothelial growth factor receptor‐2/ligand complexes by small‐angle X‐ray solution scattering

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.—Kisko, K., Brozzo, M. S., Missimer, J., Schleier, T., Menzel, A., Leppänen, V.‐M., Alitalo, K., Walzthoeni, T., Aebersold, R., Ballmer‐Hofer, K. Structural analysis of vascular endothelial growth factor receptor‐2/ligand complexes by small‐angle X‐ray solution scattering. FASEB J. 25, 2980–2986 (2011). www.fasebj.org

[1]  Pau Bernadó,et al.  Effect of interdomain dynamics on the structure determination of modular proteins by small-angle scattering , 2010, European Biophysics Journal.

[2]  D. Svergun,et al.  Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution , 2003, Quarterly Reviews of Biophysics.

[3]  R. Aebersold,et al.  Probing Native Protein Structures by Chemical Cross-linking, Mass Spectrometry, and Bioinformatics , 2010, Molecular & Cellular Proteomics.

[4]  T. C. Huang,et al.  X‐ray powder diffraction analysis of silver behenate, a possible low‐angle diffraction standard , 1993 .

[5]  F. Winkler,et al.  Crystal Structure of the Orf Virus NZ2 Variant of VEGF-E , 2006 .

[6]  K. Ballmer-Hofer,et al.  Structure and function of VEGF receptors , 2009, IUBMB life.

[7]  Lukas N. Mueller,et al.  Corrigendum: Identification of cross-linked peptides from large sequence databases , 2008, Nature Methods.

[8]  Charles Eigenbrot,et al.  Crystal Structure at 1.7 Å Resolution of VEGF in Complex with Domain 2 of the Flt-1 Receptor , 1997, Cell.

[9]  Greg L. Hura,et al.  X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. , 2011, Quarterly reviews of biophysics.

[10]  K. Ballmer-Hofer,et al.  Transmembrane domain‐mediated orientation of receptor monomers in active VEGFR‐2 dimers , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[11]  J. Schlessinger Cell Signaling by Receptor Tyrosine Kinases , 2000, Cell.

[12]  T. Jovin,et al.  Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis , 2010, Proceedings of the National Academy of Sciences.

[13]  A. Goldman,et al.  Structural determinants of growth factor binding and specificity by VEGF receptor 2 , 2010, Proceedings of the National Academy of Sciences.

[14]  O. Mayans,et al.  A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain , 2008, Proceedings of the National Academy of Sciences.

[15]  Ruedi Aebersold,et al.  Identification of cross-linked peptides from large sequence databases , 2008, Nature Methods.

[16]  Kurt Ballmer-Hofer,et al.  Crystal Structure of the Orf Virus NZ2 Variant of Vascular Endothelial Growth Factor-E , 2006, Journal of Biological Chemistry.

[17]  D. Svergun,et al.  Structural characterization of proteins and complexes using small-angle X-ray solution scattering. , 2010, Journal of structural biology.

[18]  M. Steinmetz,et al.  Structure of a VEGF–VEGF receptor complex determined by electron microscopy , 2007, Nature Structural &Molecular Biology.

[19]  J. Schlessinger,et al.  Signaling by Receptor Tyrosine Kinases , 1993 .

[20]  A. Nash,et al.  Crystal structure of human vascular endothelial growth factor-B: identification of amino acids important for receptor binding. , 2006, Journal of molecular biology.

[21]  Joseph Schlessinger,et al.  Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor , 2007, Cell.

[22]  J. Schlessinger,et al.  Contacts between membrane proximal regions of the PDGF receptor ectodomain are required for receptor activation but not for receptor dimerization , 2008, Proceedings of the National Academy of Sciences.

[23]  A. D. de Vos,et al.  Vascular endothelial growth factor: crystal structure and functional mapping of the kinase domain receptor binding site. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[24]  K. Alitalo,et al.  Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization. , 2010, Cancer cell.

[25]  A. D. de Vos,et al.  The Crystal Structure of Placental Growth Factor in Complex with Domain 2 of Vascular Endothelial Growth Factor Receptor-1* , 2004, Journal of Biological Chemistry.

[26]  K. Alitalo,et al.  Structural determinants of vascular endothelial growth factor-D receptor binding and specificity. , 2011, Blood.

[27]  R. Jaussi,et al.  Vascular Endothelial Growth Factor (VEGF) and Its Receptors in Tumor-Bearing Dogs , 1999, Biological chemistry.

[28]  G. J. Swaminathan,et al.  The Crystal Structure of Human Placenta Growth Factor-1 (PlGF-1), an Angiogenic Protein, at 2.0 Å Resolution* , 2001, The Journal of Biological Chemistry.

[29]  Peter V. Konarev,et al.  ATSAS 2.1 – towards automated and web-supported small-angle scattering data analysis , 2007 .

[30]  Pablo Chacón,et al.  Using Situs for the registration of protein structures with low-resolution bead models from X-ray solution scattering , 2001 .

[31]  Derek Toomre,et al.  Spatial control of EGF receptor activation by reversible dimerization on living cells , 2010, Nature.

[32]  J. Schlessinger,et al.  Direct contacts between extracellular membrane-proximal domains are required for VEGF receptor activation and cell signaling , 2010, Proceedings of the National Academy of Sciences.

[33]  Friedrich Förster,et al.  Structure of the 26S proteasome from Schizosaccharomyces pombe at subnanometer resolution , 2010, Proceedings of the National Academy of Sciences.