Crystal Structure of the Orf Virus NZ2 Variant of Vascular Endothelial Growth Factor-E

Abstract

Mammalian vascular endothelial growth factors constitute a family of polypeptides, vascular endothelial growth factor (VEGF)-A, -B, -C, -D and placenta growth factor (PlGF), that regulate blood and lymphatic vessel development. VEGFs bind to three types of receptor tyrosine kinases, VEGF receptors 1, 2, and 3, that are predominantly expressed on endothelial and some hematopoietic cells. Pox viruses of the Orf family encode highly related proteins called VEGF-E that show only 25-35% amino acid identity with VEGF-A but bind with comparable affinity to VEGFR-2. The crystal structure of VEGF-E NZ2 described here reveals high similarity to the known structural homologs VEGF-A, PlGF, and the snake venoms Vammin and VR-1, which are all homodimers and contain the characteristic cysteine knot motif. Distinct conformational differences are observed in loop L1 and particularly in L3, which contains a highly flexible GS-rich motif that differs from all other structural homologs. Based on our structure, we created chimeric proteins by exchanging selected segments in L1 and L3 with the corresponding sequences from PlGF. Single loop mutants did not bind to either receptor, whereas a VEGF-E mutant in which both L1 and L3 were replaced gained affinity for VEGFR-1, illustrating the possibility to engineer receptor-specific chimeric VEGF molecules. In addition, changing arginine 46 to isoleucine in L1 significantly increased the affinity of VEGF-E for both VEGF receptors.

Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization

“…L1 and L3 show the largest differences, both in conformation and in sequence, of all of the structural elements of VEGFR-2 specific ligands. Our data also clearly show that ligand structure is modulated by receptor binding; for example, L3 of VEGF-E, which is flexible in the unbound form, 15 adopts an extended ␤-sheet conformation apparently required for optimal interaction with D2 and D3. Combining the structural information now available for all 3 complexes with the analysis of the VEGF-A alanine mutant screen will be helpful in designing small-receptor-inhibitory polypeptides with therapeutic potential.…”

“…It is highly flexible in unbound VEGF-E and adopts an extended ␤-sheet conformation after receptor binding. 15 Finally, L1 of all VEGFs interacts solely with D3 ( Figure 5D). Our results provide the structural basis for the interaction of VEGF family proteins with VEGFR-2 and define subtle but distinct differences in their receptor-binding mode.…”

“…[13][14][15][16][17][18] In addition, the structures of complexes between VEGF-A, VEGF-B, and PlGF with VEGFR-1 Ig domain 2 (D2) [19][20][21] and of VEGFR-2 D23 in complex with VEGF-C are available. 14 These structures show that high-affinity binding of VEGF to VEGFR-1 arises from interaction with D2, whereas VEGFR-2 binding requires both domains D2 and D3.…”

Structural determinants of growth factor binding and specificity by VEGF receptor 2

“…2E). Moreover, Glu169, which is highly conserved in the VEGF family (29), forms a salt bridge with VEGFR-2 Lys286 and a hydrogen bond with the main chain amide of VEGFR-2 Asn253, and VEGF-C Asp123 is in contact with the D2 Arg164 and Tyr165. The site 2 VEGF-C interface (Fig.…”

“…Crystal structures have been published for VEGF-A (26), PlGF (27), VEGF-B (28), and VEGF-E (29). In addition, structures for VEGF-A (30) and PlGF (31) in complex with domain 2 of VEGFR-1 (VEGFR-1D2) are available.…”