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 ofVEGF-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.

High-resolution structures of a heterochiral coiled coil

“…In one of these systems (32), native-like quaternary contacts are maintained. In the other two examples (8,41), there is no chiral crystal structure to which direct comparison could be made; however, indirect evidence suggests in both cases that quaternary associations observed in the solid state are comparable to those that occur in solution. The structures reported here, on the other hand, show that the native tetrameric assembly of homochiral M2-TM is not maintained when the racemate is crystallized.…”

“…The available set of racemic and quasiracemic protein crystal structures allows 14 comparisons with chiral crystal counterparts (8, 29, 32, 33, 35-39, 42, 43), and all seem to suggest that native tertiary structure is maintained. Only three racemate structures include specific native-like quaternary contacts (8,32,41). In one of these systems (32), native-like quaternary contacts are maintained.…”

“…"Mirror-image phage display," a powerful method pioneered by Kim and co-workers, has identified D peptides that bind tightly to L proteins (6). A few of these heterochiral complexes have been characterized at atomic resolution (7,8), but no general principles of heterochiral recognition have emerged. Shai and co-workers have reported that D peptides corresponding to transmembrane helices of several integral membrane proteins can pair with the natural L-peptide helix within a lipid bilayer (9-11).…”

Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity

“…5), the production of larger synthetic D-proteins is becoming increasingly feasible [e.g., 204-residue D-VEGF dimer (6) and 84-residue D-MDM2/MDMX (7)]. However, many proteins are prone to misfolding, especially as their size and complexity increase (8).…”

“…Due to great interest in mirror-image proteins as targets for drug discovery (6,7,18,19) and mirror-image synthetic biology (20,21), we were intrigued by the possibility that natural (L-) GroEL/ES could assist in the folding of D-proteins. Thus, we synthesized a D-version of a substrate protein and evaluated its folding by GroEL/ES.…”