Structural basis for VEGF-C binding to neuropilin-2 and sequestration by a soluble splice form

Abstract

Vascular endothelial growth factor C (VEGF-C) is a potent lymphangiogenic cytokine that signals via the coordinated action of two cell surface receptors, Neuropilin-2 (Nrp2) and VEGFR-3. Diseases associated with both loss and gain of VEGF-C function, lymphedema and cancer, respectively, motivate studies of VEGF-C/Nrp2 binding and inhibition. Here, we demonstrate that VEGF-C binding to Nrp2 is regulated by C-terminal proteolytic maturation. The structure of the VEGF-C C terminus in complex with the ligand binding domains of Nrp2 demonstrates that a cryptic Nrp2 binding motif is released upon proteolysis, allowing specific engagement with the b1 domain of Nrp2. Based on the identified structural requirements for Nrp2 binding to VEGF-C, we hypothesized that the endogenous secreted splice form of Nrp2, s9Nrp2, may function as a selective inhibitor of VEGF-C. We find that s9Nrp2 forms a stable dimer that potently inhibits VEGF-C/Nrp2 binding and cellular signaling. These data provide critical insight into VEGF-C/Nrp2 binding and inhibition.

Figure 1. Crystal structure of the VEGF-C/Nrp2 complex reveals the basis for proteolytic-dependent binding

(A) Organization of the VEGF-C pro-protein and site of C-terminal processing (black arrow). (B) Peptides corresponding to processed (green circle) and unprocessed (black triangle) VEGF-C were assayed for the ability to bind Nrp2-b1b2 as measured by DSF thermal shift assay. Peptides were added to Nrp2-b1b2 to a final concentration of 0.5 mM and melting was monitored between 20 and 90°C. All samples were measured in triplicate and a representative melting curve is shown for each. (C) Processed VEGF-C dose-dependently enhances the Nrp2-b1b2 T_m. Error bars indicate the standard deviation (SD) of the three measurements. (D) Structure of Nrp2-b1b2 (blue) in complex with the C-terminus of VEGF-C (green). (E) Cross-section of the Nrp2 binding pocket demonstrates that the free carboxy terminus of VEGF-C is buried against the Nrp2 C-wall, which is formed by the third coagulation factor loop.

Figure 2. Mechanism of VEGF-C binding to Nrp2

(A) Zoom of the intermolecular interface between Nrp2 (blue) and VEGF-C (green) with the 2F_o-F_c electron density map for VEGF-C contoured at 1.0σ. Interfacing water is shown as grey spheres. (B) Ligplot+ generated representation of the interaction between VEGF-C (green) and Nrp2 (blue). Bond distances (Å) are labeled in black and water is shown as grey spheres. (C) Nrp2 binding was compared between VEGF-C and VEGF-C R223E. Binding was measured in triplicate and is reported as mean ± SD (*p<0.05). (D) Superimposition of the VEGF-A HBD/Nrp1 complex (PDB=4DEQ) and the tuftsin/Nrp1 complex (PDB=2ORZ) onto the structure of the VEGF-C/Nrp2 complex demonstrates the shared and unique modes of engagement within this ligand/receptor family.

Figure 3. Crystal structure and VEGF-C binding properties of Nrp2-T319R

(A) Structure of Nrp2-T319R with the stick representation for T319R shown in red. (B) Zoom of the Nrp2-T319R binding pocket. The blue mesh illustrates the 2F_o-F_c electron density map for R319 contoured at 1.0σ. (C) Superimposition of VEGF-C (green) onto the structure of Nrp2-T319R demonstrates that the binding pocket normally occupied by VEGF-C is blocked in the mutant. (D) VEGF-C binding was compared between Nrp2-b1b2 and Nrp2-T319R. Binding was measured in triplicate and is reported as mean ± SD (*p<0.05).

Figure 4. s₉Nrp2^B forms a disulfide-linked dimer

(A) Domain organization of Nrp2 and the protein fragment utilized for our studies, s₉Nrp2^B. (B) Western blot analysis of Hgh-tagged s₉Nrp2 and s₉Nrp2^B expressed in CHO cells. (C) Non-reducing and reducing SDS-PAGE analysis of Nrp2-b1 and s₉Nrp2^B. (D) The oligomeric state of s₉Nrp2^B (black line) was analyzed by size-exclusion chromatography. Nrp2-b1 was run as a reference (grey line).

Figure 5. Crystal structure and inhibitory properties of s₉Nrp2^B

(A) Crystal structure of the s₉Nrp2^B dimer (Chain A: light orange; Chain B: dark orange). The intermolecular disulfide is shown in black and the Nrp2-b1 binding pockets are labeled with arrows. (B) Zoom of the dimeric helical bundle with the 2F_o-F_c electron density map contoured at 1σ. (C) The residues of the Nrp2 b1-b2 linker and b2 domain show a dramatic structural reorganization from an extended loop in the b1b2 sequence (blue) to an extended helix in the s₉Nrp2^B dimer (orange). (D) ATWLPPR (grey), Nrp2-b1 (blue), and s₉Nrp2^B (orange) were assayed for the ability to inhibit VEGF-C binding to Nrp2. ATWLPPR inhibited binding with an IC₅₀ = 10 μM (log[IC₅₀] = −4.98 ± 0.03), Nrp2-b1 inhibited binding with an IC₅₀ = 1.5 μM (log[IC₅₀] = −5.82 ± 0.09), and s₉Nrp2^B inhibited binding with an IC₅₀ = 250 nM (log[IC₅₀] = −6.60 ± 0.08). (E) s₉Nrp2^B was assayed for the ability to alter VEGF-C binding to VEGFR3. Addition of 4μM s₉Nrp2^B fully inhibited VEGF-C/Nrp2 binding but showed no effect on VEGF-C/VEGFR3 binding. (F) Inhibition of C4-2 cell prostatosphere formation was used to assess the biological activity of s₉Nrp2^B. Prostatosphere formation was compared in the absence and presence of s₉Nrp2^B, as well as with C-furSema (positive control) and C-Sema (negative control). (G) Model illustrating the mechanism of action for s₉Nrp2^B. s₉Nrp2^B sequesters VEGF-C and prevents activation of the VEGFR3/Nrp2 signaling complex. All inhibition experiments were measured in triplicate and reported as mean ± SD (*p<0.05).