title: Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography.
abstract: Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637, USA.
authors.forename: Kalyaneswar
authors.initials: K
authors.lastname: Mandal
authors.forename: Maruti
authors.initials: M
authors.lastname: Uppalapati
authors.forename: Dana
authors.initials: D
authors.lastname: Ault-Riché
authors.forename: John
authors.initials: J
authors.lastname: Kenney
authors.forename: Joshua
authors.initials: J
authors.lastname: Lowitz
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.forename: Stephen B H
authors.initials: SB
authors.lastname: Kent
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D008024:Ligands; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D016366:Open Reading Frames; D013237:Stereoisomerism; D020349:Surface Plasmon Resonance; D042461:Vascular Endothelial Growth Factor A / Q000138:chemical synthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D008024:Ligands; D046912:Multiprotein Complexes; D042461:Vascular Endothelial Growth Factor A