Mechanism of Selective VEGF-A Binding by Neuropilin-1 Reveals a Basis for Specific Ligand Inhibition
Figure 2
Nrp1 residues mediate specific VEGF-A binding.
(A) Alignment of orthologous Nrp1 and Nrp2 b1 domains shows conservation of residues critical for C-terminal arginine binding (marked with a *) but variability within regions surrounding the interloop cleft (orange: Nrp1:E285/Nrp2:R287 [9]; green: Nrp1∶299-TN-300/Nrp2∶301-DGR-303; blue: Nrp1∶304-ER-305/Nrp2∶307-QQ-308; purple: Nrp1∶350-KKK-352/Nrp2∶353-QNG-355). Below the alignment is a conservation histogram illustrating identity across the displayed sequences. (B) Surface representation of the Nrp b1 domain reveals that the direct VEGF-A binding region (gold) [16] is closely associated with the selected regions (colored according to 2A) in three-dimensional space. (C) VEGF-A binding of Nrp1 mutants reveals loss of binding for each mutant protein compared to wild-type. Retained AP-Nrp1 binding is reported as the percent of retained wild-type AP-Nrp1. (D) Determination of the VEGF-A binding capacity of Nrp2 mutants reveals that three of the four Nrp2 chimeras show enhanced VEGF-A binding compared to wild-type. Retained AP-Nrp2 binding is reported as the percent of retained wild-type AP-Nrp2. Experiments were performed in triplicate and reported as the mean ±1 S.D.