Structural basis for ligand and heparin binding to neuropilin B domains

Neuropilin (Nrp) is a cell surface receptor with essential roles in angiogenesis and axon guidance. Interactions between Nrp and the positively charged C termini of its ligands, VEGF and semaphorin, are mediated by Nrp domains b1 and b2, which share homology to coagulation factor domains. We report here the crystal structure of the tandem b1 and b2 domains of Nrp-1 (N1b1b2) and show that they form a single structural unit. Cocrystallization of N1b1b2 with Tuftsin, a peptide mimic of the VEGF C terminus, reveals the site of interaction with the basic tail of VEGF on the b1 domain. We also show that heparin promotes N1b1b2 dimerization and map the heparin binding site on N1b1b2. These results provide a detailed picture of interactions at the core of the Nrp signaling complex and establish a molecular basis for the synergistic effects of heparin on Nrp-mediated signaling.

[1]  B. Keyt,et al.  Crystallization of the receptor binding domain of vascular endothelial growth factor , 1996, Proteins.

[2]  L Holm,et al.  Alignment of three-dimensional protein structures: network server for database searching. , 1996, Methods in enzymology.

[3]  S. Crennell,et al.  The three domains of a bacterial sialidase: a beta-propeller, an immunoglobulin module and a galactose-binding jelly-roll. , 1995, Structure.

[4]  S. Soker,et al.  Identification of a natural soluble neuropilin-1 that binds vascular endothelial growth factor: In vivo expression and antitumor activity. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Wayne A. Hendrickson,et al.  Structure of a heparin-linked biologically active dimer of fibroblast growth factor , 1998, Nature.

[6]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[7]  J. Wood,et al.  A VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding shows attenuated signaling through VEGFR-2 , 2006, Cellular and Molecular Life Sciences CMLS.

[8]  M. Shibuya,et al.  Neuropilin-1 Is a Placenta Growth Factor-2 Receptor* , 1998, The Journal of Biological Chemistry.

[9]  K. Fujikawa,et al.  New insights into binding interfaces of coagulation factors V and VIII and their homologues lessons from high resolution crystal structures. , 2002, Current protein & peptide science.

[10]  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.

[11]  G. Neufeld,et al.  The neuropilins and their role in tumorigenesis and tumor progression. , 2006, Cancer letters.

[12]  Kharitonov Sv,et al.  The psychophysiology of neuroses , 1999 .

[13]  Thomas Terwilliger,et al.  SOLVE and RESOLVE: automated structure solution, density modification and model building. , 2004, Journal of synchrotron radiation.

[14]  Chenghua Gu,et al.  Characterization of Neuropilin-1 Structural Features That Confer Binding to Semaphorin 3A and Vascular Endothelial Growth Factor 165* , 2002, The Journal of Biological Chemistry.

[15]  A. Cardin,et al.  Molecular Modeling of Protein‐Glycosaminoglycan Interactions , 1989, Arteriosclerosis.

[16]  J. Schlessinger,et al.  Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. , 2000, Molecular cell.

[17]  W. Arap,et al.  Structural basis for the interaction of a vascular endothelial growth factor mimic peptide motif and its corresponding receptors. , 2005, Chemistry & biology.

[18]  L. Ellis.  The role of neuropilins in cancer , 2006, Molecular Cancer Therapeutics.

[19]  D. Guidolin,et al.  Loss of inhibitory semaphorin 3A (SEMA3A) autocrine loops in bone marrow endothelial cells of patients with multiple myeloma. , 2006, Blood.

[20]  Jay Painter,et al.  Electronic Reprint Biological Crystallography Optimal Description of a Protein Structure in Terms of Multiple Groups Undergoing Tls Motion Biological Crystallography Optimal Description of a Protein Structure in Terms of Multiple Groups Undergoing Tls Motion , 2005 .

[21]  H. Cai,et al.  Cloning and Characterization of Neuropilin-1-Interacting Protein: A PSD-95/Dlg/ZO-1 Domain-Containing Protein That Interacts with the Cytoplasmic Domain of Neuropilin-1 , 1999, The Journal of Neuroscience.

[22]  K. Garcia,et al.  The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1. , 2000, The Journal of biological chemistry.

[23]  K. Agata,et al.  The A5 antigen, a candidate for the neuronal recognition molecule, has homologies to complement components and coagulation factors , 1991, Neuron.

[24]  M. Tessier-Lavigne,et al.  Neuropilin Is a Receptor for the Axonal Chemorepellent Semaphorin III , 1997, Cell.

[25]  K. Fujikawa,et al.  Structure of the C2 domain of human factor VIII at 1.5 Å resolution , 1999, Nature.

[26]  A. Weston,et al.  Overexpression of vascular endothelial growth factor164 and its co-receptor neuropilin-1 in estrogen-induced rat pituitary tumors and GH3 rat pituitary tumor cells. , 2000, International journal of oncology.

[27]  G. Spraggon,et al.  Crystal structure of the human neuropilin-1 b1 domain. , 2003, Structure.

[28]  H. Fujisawa,et al.  Determination of Cell Adhesion Sites of Neuropilin-1 , 2000, The Journal of cell biology.

[29]  M. Crépin,et al.  Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1. , 2006, Life sciences.

[30]  O. Cussenot,et al.  VEGF overexpression in clinically localized prostate tumors and neuropilin‐1 overexpression in metastatic forms , 2000, International journal of cancer.

[31]  S. Soker,et al.  Inhibition of Vascular Endothelial Growth Factor (VEGF)-induced Endothelial Cell Proliferation by a Peptide Corresponding to the Exon 7-Encoded Domain of VEGF165 * , 1997, The Journal of Biological Chemistry.

[32]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[33]  M. Hori,et al.  Glycosaminoglycan modification of neuropilin‐1 modulates VEGFR2 signaling , 2006, The EMBO journal.

[34]  M. Klagsbrun,et al.  Neuropilins in neoplasms: expression, regulation, and function. , 2006, Experimental cell research.

[35]  Sushanta K Banerjee,et al.  Neuropilin‐1 is differentially expressed in myoepithelial cells and vascular smooth muscle cells in preneoplastic and neoplastic human breast: A possible marker for the progression of breast cancer , 2002, International journal of cancer.

[36]  P. Driscoll,et al.  Characterization of a Bicyclic Peptide Neuropilin-1 (NP-1) Antagonist (EG3287) Reveals Importance of Vascular Endothelial Growth Factor Exon 8 for NP-1 Binding and Role of NP-1 in KDR Signaling* , 2006, Journal of Biological Chemistry.

[37]  N. Ferrara,et al.  The biology of VEGF and its receptors , 2003, Nature Medicine.

[38]  Zhigang He,et al.  Semaphorin–Neuropilin Interactions Underlying Sympathetic Axon Responses to Class III Semaphorins , 1998, Neuron.

[39]  Nijole Gasiunas,et al.  Neuropilin-1 Binds Vascular Endothelial Growth Factor 165, Placenta Growth Factor-2, and Heparin via Its b1b2 Domain* , 2002, The Journal of Biological Chemistry.

[40]  M. Klagsbrun,et al.  The role of neuropilin in vascular and tumor biology. , 2002, Advances in experimental medicine and biology.

[41]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[42]  M. Kreuter,et al.  Correlation of neuropilin-1 overexpression to survival in acute myeloid leukemia , 2006, Leukemia.

[43]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[44]  M. Schachner,et al.  Analysis of the L1-Deficient Mouse Phenotype Reveals Cross-Talk between Sema3A and L1 Signaling Pathways in Axonal Guidance , 2000, Neuron.

[45]  D. Goeddel,et al.  Vascular endothelial growth factor is a secreted angiogenic mitogen. , 1989, Science.

[46]  A. Vagin,et al.  MOLREP: an Automated Program for Molecular Replacement , 1997 .

[47]  Christopher J. Robinson,et al.  VEGF165-binding Sites within Heparan Sulfate Encompass Two Highly Sulfated Domains and Can Be Liberated by K5 Lyase* , 2006, Journal of Biological Chemistry.

[48]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[49]  Anastassis Perrakis,et al.  Developments in the CCP4 molecular-graphics project. , 2004, Acta crystallographica. Section D, Biological crystallography.

[50]  S. Kenwrick,et al.  Cis and trans interactions of L1 with neuropilin‐1 control axonal responses to semaphorin 3A , 2002, The EMBO journal.

[51]  L. Ellis,et al.  Neuropilin-1 in human colon cancer: expression, regulation, and role in induction of angiogenesis. , 2004, The American journal of pathology.

[52]  Alex L Kolodkin,et al.  Neuropilin Is a Semaphorin III Receptor , 1997, Cell.

[53]  Alex L Kolodkin,et al.  Neuropilin-2 Is a Receptor for Semaphorin IV Insight into the Structural Basis of Receptor Function and Specificity , 1998, Neuron.

[54]  M. Devocelle,et al.  A peptide corresponding to the neuropilin-1-binding site on VEGF165 induces apoptosis of neuropilin-1-expressing breast tumour cells , 2005, British Journal of Cancer.

[55]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[56]  E. Marinelli,et al.  Tuftsin Binds Neuropilin-1 through a Sequence Similar to That Encoded by Exon 8 of Vascular Endothelial Growth Factor* , 2006, Journal of Biological Chemistry.

[57]  M. Pop,et al.  An optimized system for expression and purification of secreted bacterial proteins. , 2006, Protein expression and purification.

[58]  B. Keyt,et al.  Solution structure of the heparin-binding domain of vascular endothelial growth factor. , 1998, Structure.

[59]  Shay Soker,et al.  Neuropilin-1 Is Expressed by Endothelial and Tumor Cells as an Isoform-Specific Receptor for Vascular Endothelial Growth Factor , 1998, Cell.

[60]  Hung V. Nguyen,et al.  The Carboxyl-terminal Domain(111165) of Vascular Endothelial Growth Factor Is Critical for Its Mitogenic Potency (*) , 1996, The Journal of Biological Chemistry.