Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization

Abstract

VEGFs activate 3 receptor tyrosine kinases, VEGFR-1, VEGFR-2, and VEGFR-3, promoting angiogenic and lymphangiogenic signaling. The extracellular receptor domain (ECD) consists of 7 Ig-homology domains; domains 2 and 3 (D23) represent the ligandbinding domain, whereas the function of D4-7 is unclear. Ligand binding promotes receptor dimerization and instigates transmembrane signaling and receptor kinase activation. In the present study, isothermal titration calorimetry showed that the Gibbs free energy of VEGF-A, VEGF-C, or VEGF-E binding to D23 or the full-length ECD of VEGFR-2 is dominated by favorable entropic contribution with enthalpic penalty. The free energy of VEGF binding to the ECD is 1.0-1.7 kcal/mol less favorable than for binding to D23. A model of the VEGF-E/ VEGFR-2 ECD complex derived from smallangle scattering data provided evidence for homotypic interactions in D4-7. We also solved the crystal structures of complexes between VEGF-A or VEGF-E with D23, which revealed comparable binding surfaces and similar interactions between the ligands and the receptor, but showed variation in D23 twist angles. The energetically unfavorable homotypic interactions in D4-7 may be required for re-orientation of receptor monomers, and this mechanism might prevent ligand-independent activation of VEGFR-2 to evade the deleterious consequences for blood and lymph vessel homeostasis arising from inappropriate receptor activation. (Blood. 2012;119(7):1781-1788) IntroductionA plethora of growth factors, such as angiopoietins, VEGF family ligands, platelet-derived growth factors, fibroblast growth factors, and hepatocyte growth factors regulate blood and lymph vessel formation and homeostasis (reviewed in Cao 1 ). VEGFs represent a large family of ligands: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and PlGF, which bind to and activate in a combinatorial fashion 3 type V receptor tyrosine kinases (RTKs), VEGFR-1, VEGFR-2, and VEGFR-3, which give rise to highly specific signal output. In mammals, VEGF-A signaling through VEGFR-2 is the major angiogenic signaling pathway, but VEGF-C plays essential, and in some cases complementary, roles in the activation of this receptor (reviewed in Grünewald et al 2 ). The mechanism by which VEGFRs are activated is not understood in molecular detail, but clearly represents one of the many variations of RTK activation. In general, signaling by RTKs requires ligand-mediated dimerization with precise positioning of receptor subunits in active dimers. Dimeric ligand/receptor complexes subsequently initiate transmembrane signaling, resulting in the activation of the intracellular tyrosine kinase domains. 3,4 Active VEGFRs instigate cell signaling and promote endothelial cell migration and proliferation, as well as vessel fenestration and permeabilization. 5,6 The extracellular domain (ECD) of VEGFRs consists of 7 Ig-homology domains. The first 3 domains mediate ligand binding, 7,8 whereas the membrane proximal domains are involved in ligand-induced receptor dimerization. 7,9 Homotypic receptor i...

Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation

“…Similarly, comparison of the available ligand/VEGFR-2 D2-3 complex structures found large variations in the D2-3 twist angles, indicating a D2-3 hinge-like rigid body motion (18,30). Taken together with our data, these findings suggest a general mechanism for VEGFR activation in which ligand-induced D2-3 reorientation facilitates homotypic interactions in D5 and D7, resulting in specific positioning of the transmembrane and intracellular kinase domains in active receptor dimers (Fig.…”

“…Consistent with the D2-3 reorientation, VEGFR-3 D3 also increases VEGF-C binding, whereas D4-5 and D6-7 further increase VEGFR-3 ligand-binding affinity. In contrast, the homotypic interactions in VEGFR-2 D4-7 are energetically unfavorable (30), suggesting that D3's contribution to ligand binding is essential for VEGFR-2 dimerization and activation. VEGFR-1 D4-7 has only a small positive effect on ligand binding (16,28).…”

“…So far, KIT seems to be the only type III/V RTK that directly uses D1 for ligand binding (22). D1 may have a positive impact on D2 structural stability, as demonstrated by the poor resolution of the first strand of D2 (βA) in the multiple VEGFR-2 D2-3 structures, which lack D1 (18,30). On the other hand, VEGFR-3 D1 has large disordered regions in the ligand complex, suggesting that it may interact with other binding partners in vivo, such as neuropilin-2 or the large N-and C-terminal propeptides of VEGF-C and VEGF-D.…”

Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

“…1B). We have recently demonstrated that homotypic receptor contacts are enthalpically unfavorable and reduce the overall binding activity of the ligand for VEGFR-2 (6). Taken together, these data suggest that the two monomers comprising the active receptor complex are held together by ligand binding to Ig domains 2 and 3 (D23) and by homotypic receptor contacts in D4 to D7 of the ECD.…”

“…We have shown recently that receptor dimerization is necessary but not sufficient for VEGFR-2 kinase activation (7), suggesting that precise orientation of receptor monomers in active dimers is critical to the instigation of transmembrane signaling. In addition, biochemical data (9,24) and high-resolution structural information for VEGF ligand/receptor complexes (6,17) revealed that extracellular immunoglobulin homology domains (Ig domains) D2 and D3 (D23) comprise the ligand binding site. Furthermore, structural information derived from electron microscopy (EM) (22) and smallangle X-ray scattering (SAXS) data (14) suggests that the ligandbound VEGFR-2 ECD is also engaged in homotypic contacts between Ig domains D4 and D7.…”