Relationship between Structural Flexibility and Function in the C-Terminal Region of the Heparin-Binding Domain of VEGF₁₆₅

Abstract

Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Previously, we reported that triamterene (Trm) inhibits VEGF-amyloid β (Aβ) interactions without affecting other biological activities of VEGF or Aβ [Jeong, K.-W., et al. (2011) Biochemistry 50, 4843-4854]. We further showed that molecular motions in the N-terminal disordered loop region of the heparin-binding domain (HBD) are important for interaction with Trm. To investigate the importance of motion at the C-terminal domain of HBD, we constructed a binding model of HBD with heparin octasaccharide (HOS) based on measurements of chemical shift changes and docking studies. Furthermore, the dynamic properties of the HBD-HOS and HBD-Trm-HOS complexes were assessed by measuring spin relaxation rates. The results showed that the HOS-binding site is composed of two basic clusters consisting of side chains of residues R13, R14, and K15 and residues K30, R35, and R49. When HOS binds, values for the heteronuclear nuclear Overhauser effect near HOS-binding sites increased dramatically. CPMG (Carr-Purcell-Meiboom-Gill sequence) experiments as well as an R2 relaxation experiment were undertaken to understand millisecond time-scale motions in HBD. There is large relaxation dispersion of residues at Trm- and HOS-binding sites in free HBD. C-Terminal residues such as S34, C48, and D51 near the HOS-binding sites continued to exhibit slow conformational motions in the HBD-Trm complex, while those slow motions disappeared in the bound conformation of HBD with HOS. Collectively, our results demonstrate that the inherent structural flexibilities of the C-terminal region of the HBD are important in the heparin binding process and that Trm does not inhibit VEGF-heparin interactions necessary for the biological activities of VEGF.

Pharmacology of Heparin and Related Drugs

“…Interactions predicted involve N-, 2-O-, and 6-O-sulfates (Robinson et al, 2006;Jeong et al, 2013) PDGF-AA Filter-trapping and affinity chromatography 6-8 monosaccharides minimum, NS domains containing both 2-O-and 6-O-sulfate groups (Feyzi et al, 1997) HGF/SF X-ray crystallography of NK1 domain with tetrasaccharide (1GMN.pdb, 1GMO.pdb) Binding to N domain critical for biologic activity (Lietha et al, 2001) Chemokines Gro-a (CXCL1) NMR of trapped dimer; titrations with octasaccharide Heparin binds orthogonally to interhelical axis of disulphide-trapped dimer; octasaccharide is not long enough to span completely both heparin binding sites in the dimer (Poluri et al, 2013).…”

“…Growth Factors. The heparin binding site of VEGF forms a belt-like region in the C-terminal domain of the HS-binding isoform of the protein (Robinson et al, 2006;Krilleke et al, 2007;Jeong et al, 2013). Angiogenesis is guided by VEGF held in the ECM by HS proteoglycans (Zoeller et al, 2009); this interaction, and its disruption by heparanase, is important for development, wound healing, and cancer (Vlodavsky et al, 2011).…”

Combination therapy with low molecular weight heparin and Adriamycin results in decreased breast cancer cell metastasis in C3H mice

“…VEGF has a major role in angiogenesis by acting via tyrosine kinase receptors. VEGF antagonists affect tumor growth and vascularization, and the VEGF-specific antibody bevacizumab exerts antivascular effects in patients with cancer (22). LMWH may also inhibit the hyperplasia of endothelial cells and competitively bind to the heparin acceptor, thus affecting angiogenesis factors, particularly VEGF (13,23).…”

Peptide Inhibitors of Vascular Endothelial Growth Factor A: Current Situation and Perspectives

“…It was shown that the minimum size among the heparin oligosaccharides, which were able to bind VEGFA 165 , was an octasaccharide. Tetradecasaccharide was able to bind VEGFA 165 with an affinity comparable to that of heparin ( Figure 3 ) [ 57 ].…”