The ability to design proteins with high affinity and selectivity for any given small molecule is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition. Attempts to rationally design ligand-binding proteins have met with little success, however, and the computational design of protein–small-molecule interfaces remains an unsolved problem. Current approaches for designing ligand-binding proteins for medica and biotechnological uses rely on raising antibodies against a target antigen in immunized animals and/or performing laboratory-directed evolution of proteins with an existing low affinity for the desired ligand, neither of which allows complete control over the interactions involved in binding.
Tinberg and Sagar D. Khare headed the study under the direction of David Baker, UW professor of biochemistry and Howard Hughes Medical Institute investigator, describe in a recent Nature paper a general computational method for designing pre-organized and shape complementary small-molecule-binding sites, and use it to generate protein binders to the steroid digoxigenin (DIG). Of seventeen experimentally characterized designs, two bind DIG; the model of the higher affinity binder has the most energetically favourable and pre-organized interface in the design set. A comprehensive binding-fitness landscape of this design, generated by library selections and deep sequencing, was used to optimize its binding affinity to a picomolar level, and X-ray co-crystal structures of two variants show atomic-level agreement with the corresponding computational models. The optimized binder is selective for DIG over the related steroids digitoxigenin, progesterone and β-oestradiol, and this steroid binding preference can be reprogrammed by manipulation of explicitly designed hydrogen-bonding interactions. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics and diagnostics.
The scientific team overcame previously unsolved problems in building accurate protein-small molecule interfaces. Earlier attempts struggled with discrepancies between the computer plans and the structures of the actual molecules.
In conducting the study, the researchers learned general principles for engineering small molecule-binding proteins with strong attraction energies. Their findings open up the possibility that binding proteins could be created for many medical, industrial and environmental uses.
In medical diagnostics, for example, a rationally programmed protein might detect biomolecules found only in a specific disease state, such as an early-stage cancer. Other types of protein molecules might eventually be manufactured to treat an overdose or to block a poison. Remediation possibilities for these molecular workhorses could include trapping pollutants or capturing waste.
Tinberg explained that generation of novel small-molecule binding proteins currently consists of immunizing an animal to generate antibodies against a target protein, or directing the evolution of proteins in a laboratory to strengthen their affinity for the desired small-molecule.
“Neither of these methods allows complete control over the interactions involved in binding,” she said.
In designing their molecules, the team sought to replicate properties of a naturally occurring protein binding site. These are: specific interactions that enforce a strong attraction with the desired small molecule, a receptive shape to accept the small molecule, and an orderly structure, prepared for occupancy. The exclusive, move-in ready set up reduces the energy penalty by preventing the protein from having to change shape to accept the small molecule. This is in contrast to a flexible site, which is more disordered in the absence of the small molecule and has to freeze into one state upon binding.