In Vitro Evolution of the Binding Specificity of Neocarzinostatin, an Enediyne-Binding Chromoprotein

Abstract

Neocarzinostatin is the most studied member of the enediyne-chromoprotein family, and is clinically used as an antitumoral agent. Neocarzinostatin could be a promising drug delivery vehicle if new binding specificities could be conferred to its protein scaffold. We used in vitro evolution methods to demonstrate that this approach is feasible. We created large libraries containing between 1.7 x 10(8) and 1.4 x 10(9) independent clones, where up to 13 side chains pointing toward the binding crevice were randomly substituted. We then used phage display to select variants that bind to a model ligand (testosterone) which is unrelated to the natural ligand of neocarzinostatin. Several different binders were selected from each library. The corresponding proteins were expressed in Escherichia coli and their affinities and specificities were characterized in detail. K(D) values of about 20 nM were obtained for streptavidin-bound testosterone. The K(D) of selected proteins for free soluble testosterone are between 7 and 55 microM and therefore higher than the K(D) for streptavidin-bound testosterone. The spacer and streptavidin used during selection contributed to the high affinity of the selected binders for the target. Binding studies of 15 different steroids related to testosterone allowed us to determine that C3, 4, 5, 6, and 7 on cycles A and B and the conjugated 3 oxo group of the steroid molecule were essential for molecular recognition. Other testosterone analogues substituted on C1, 2, 9, 11, 15, and 17 were not discriminated from testosterone. These results demonstrate that the binding specificity of this protein family can be extended to compounds that are completely unrelated to the natural enediyne chromophore family. This type of highly expressed, stable proteins with tailored binding properties have a wide potential range of applications.

A New Model for Ligand Release

“…stabilizing the bound drug and releasing the same by operating the side chain of a critical amino acid. apoNCS and its evolution variants are able to carry a number of small molecules such as ethidium bromide (50), nitrogen mustard (51), or testosterone (52). Taking into account of the enhanced DNA cleavage by Phe 78 mutants, the present drug release model provides a promising indication for engineering pharmacological packages in a broad functional context.…”

Solution NMR Structure Investigation for Releasing Mechanism of Neocarzinostatin Chromophore from the Holoprotein

“…However, holo-NCS is very unstable under the condition because of the free chromophore and we could not yet find a suitable condition for structure determination and relaxation studies. A possible alternative will be the investigation of the chromophore mimic compound complex with apo-NCS (11)(12)(13) in the dissociative condition with a combination of a static/dynamic structure comparison with holo-NCS. Our findings predict that an introduction of the aminosugar in the mimic compound significantly increases the binding affinity to allow the investigation under the dissociative condition.…”

“…These mechanisms were dominated by hydrophobic interactions with the binding pocket. However, the mechanism of releasing and binding of the chromophore remains unclear, even though the process has attracted much attention and has been investigated by a variety of methods (8,11,22,23). In the previous NMR structure ensemble, the loop structures have particularly poor convergence and this was assumed to be caused by structural flexibilities, which were thought to be responsible for the chromophore-releasing mechanism (15,17,18).…”

Preparation of Neocarzinostatin Apoprotein Mutants and the Randomized Library on the Chromophore-Binding Cavity

“…9) Nicaise et al reported the affinity transfer from antibody to apo-NCS by the CDR3 grafting of the VHH chain of camel anti-lysozyme antibody. Thus, apo-NCS randomized library would be one of most promising resource for newly caged protein.…”