title: Effect of C-terminal sequence on competitive semaphorin binding to neuropilin-1.
abstract: Neuropilins (Nrp) are type I transmembrane proteins that function as receptors for vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3) ligand families. Sema3s function as potent endogenous angiogenesis inhibitors but require proteolytically processing by furin to compete with VEGF for Nrp binding. This processing liberates a C-terminal arginine (CR) that is necessary for binding to the b1 domain of Nrp, a common feature shared by Nrp ligands. The CR is necessary but not sufficient for potent Nrp inhibition, and the role of upstream residues is unknown. We demonstrate that the second-to-last residue (C-1), immediately upstream of the CR, plays a significant role in controlling competitive ligand binding by orienting the C-terminus for productive Nrp binding. With the use of a peptide library derived from Sema3F, C-1 residues that preferentially adopt an extended bound-like conformation, including proline and β-branched amino acids, were found to produce the most avid competitors. Consistent with this, analysis of the binding thermodynamics revealed that more favorable entropy is responsible for the observed binding enhancement of C-1 proline. We further tested the effect of the C-1 residue on Sema3F processing by furin and found an inverse relationship between processing and inhibitory potency. Analysis of all Sema3 family members reveals two non-equivalent furin processing sites differentiated by the presence of either a C-1 proline or a C-1 arginine and resulting in up to a 40-fold difference in potency. These data reveal a novel regulatory mechanism of Sema3 activity and define a fundamental mechanism for preferential Nrp binding.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 40536, USA.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Parker
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Linkugel
authors.forename: Craig W
authors.initials: CW
authors.lastname: Vander Kooi
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001667:Binding, Competitive; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D039942:Neuropilin-1 / Q000737:chemistry* / Q000378:metabolism; D056510:Position-Specific Scoring Matrices; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D059748:Proteolysis; D039961:Semaphorins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008024:Ligands; D039961:Semaphorins; D039942:Neuropilin-1; D001120:Arginine
keywords: AP; CHO; Chinese hamster ovary; ITC; Sema3; VEGF; alkaline phosphatase; angiogenesis; class III Semaphorin; furin; isothermal titration calorimetry; peptide library; proteolysis; vascular endothelial growth factor