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doi:10.1038/nature12463

A recipe for ligandbinding proteins

Cellular cross-talk, enzymatic catalysis and regulation of gene expression all depend on molecular recognition. A method that allows the design of proteins with desired recognition sites could thus be revolutionary.
G I O VA N N A G H I R L A N D A

o support cellular processes, natural proteins have evolved to recognize a relatively small set of ligand molecules with high affinity and specificity. Broadening this set of proteinligand pairs with synthetic proteins that are specific for ligands of choice could transform the development of biosensors, protein-based drugs, artificial enzymes and tools for chemical biology. Current invitro approaches to the design of proteinligand pairs rely on laborious directed-evolution techniques to engineer binding sites in existing protein scaffolds, and cannot be generalized. In a paper published on Natures website today, Tinberg et al.1 describe a computational method that promises to streamline and greatly facilitate the development of tailored ligand-binding proteins. One way to engineer synthetic ligandbinding proteins is repurposing existing proteins through directed evolution. This approach involves selecting a few amino acids within an existing protein, typically located in a cavity, and randomly mutating them to create
a Idealized binding site

libraries of millions of protein variants, which are then selected for their ability to bind the target. Depending on the ligand and the scaffold protein, successive rounds of directed evolution yield binding proteins with dissociation constants in the physiologically relevant micromolar to nanomolar ranges2,3. The process is arduous, and success is not guaranteed: current methods limit the size of combinatorial libraries to about 109, which is often insufficient to explore a large number of positions and so limits the fitness of the resulting binders. Furthermore, the process works best when the starting protein already binds weakly to the desired ligand, rather than when starting from scratch. Computational methods hold great promise to bypass this lengthy process by using virtual selection on a large number of scaffolds. Such approaches have enjoyed much success lately, with the design of artificial enzymes for a variety of reactions48. Despite these feats, the design of high-affinity ligand-binding proteins has remained elusive9. Tinberg et al. now present a generalizable strategy for designing artificial recognition modules for any desired target molecule.
b
Two viable candidates Kd = 10 M

As their target they chose digoxigenin a natural steroid that forms part of the cardiac drug digoxin, and that has found a second life as a recognition tag in biotechnology applications. Consequently, a few digoxigeninbinding agents, including both antibodies and non-antibody proteins, have been developed through traditional protein-engineering methods1012. Taking some clues from the previously developed binding agents, Tinberg and colleagues sketched out a couple of possible ways to bind ligands to digoxigenin using amino acids that could provide hydrogen bonds and hydrophobic interactions (Fig. 1a). They then transposed these minimalistic sets into three-dimensional models, which were used as probes to interrogate a selected set of 401proteins, encompassing several classes of protein fold, for geometrical compatibility. Powerful computational methods available in the authors lab enabled them to rapidly sift through all possible matches, rank the candidates and perform optimization through rounds of redesign and scoring. Seventeen proteins emerged as candidates for experimental characterization, on the basis of optimal shape complementarity to the ligand, pre-organization of the site in the absence of ligand and predicted protein stability. Of these, only two bound the ligand with micromolar affinity (Fig. 1b). Although an impressive result for a computational approach, these binding affinities are far from the nanomolar ranges needed for practical applications. The authors, therefore, turned to directed-evolution methods to improve binding affinity. To explore the effect of all 20 possible amino acids at each of 34 residues located within
c The winner

Kd = 500 pM

Digoxigenin Hydrogen bond

Sca old matching

Directed evolution

Amino-acid side chains

Figure 1 | Steps to making a ligand-binding protein.a, To design a protein with high binding affinity for the steroid digoxigenin, Tinberg et al.1 created a minimal digoxigenin-binding site made with amino-acid side chains positioned to maximize hydrogen bonding and hydrophobic interactions to the target. They then sifted through a selected set of 401 scaffold-protein structures to find all (17) possible matches for the digoxigenin-binding site

and ranked them by best fit. b,After validating the predictions through protein expression, they subjected the two best performers, which had micromolar binding affinities (Kd), to directed-evolution methods, randomizing amino acids farther away from the binding site. c, Finally, a protein with picomolar binding affinity emerged as winner, and the authors obtained its crystal structure to confirm the design features.
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roughly 10 ngstrms of the binding site would have required an astronomical number (more than 1044) of possible protein sequences. Fortunately, a new method13 allowed Tinberg et al. to circumvent this limitation by randomizing each of the 34 positions independently, selecting for binding to labelled digoxigenin, and using deep sequencing to build a fitness map that correlates amino-acid identity at each position to function. On the basis of this information, the authors combined favourable substitutions in successive generations of libraries to obtain mutants with affinity in the lownanomolar to high-picomolar ranges (Fig. 1c). The litmus test for measuring successful protein design is obtaining high-resolution structures of the final product and comparing it with the designed features in the models. Tinberg and colleagues proteins pass this test with flying colours: structural characterization of the two best mutants, DIG10.2 and its improved version, DIG10.3, showed the presence of hydrogen bonding between three designed tyrosine residues and the ligand. Tyrosine is frequently found in the binding pockets of antibodies and of synthetic ligandbinding proteins: its ability to form hydrophobic and hydrogen-bonding inter actions, as well as its size and conformational rigidity, fulfil many roles in molecular recognition14. In addition to the designed features, subjecting the proteins to directed evolution allowed the exchange of small hydrophobic amino acids with larger ones, and the introduction of two additional tyrosines within the binding site. These mutations reinforce design principles incorporated in the computational selection, such as shape complementarity and bindingsite pre-organization, which maximize the entropic contribution to binding as well as maximizing specificity. Tinberg et al. also exploited hydrogenbonding interactions, which impart strong geometric constraints and thus contribute to specificity for binding to digoxigenin as opposed to other steroids. This point is elegantly illustrated by a series of mutants in which the authors correlate the number of hydrogen-bonding groups on the target steroids with the effect of swapping each tyrosine for phenylalanine, which cannot hydrogen bond, and demonstrate the large energetic penalty paid for each mismatch. The success of this protocol overcomes a long-standing issue in protein design, and promises to have an impact on the effectiveness of designed enzymes, which has often been limited by low affinity for the substrate. This work also opens up many exciting challenges. Digoxigenin proved a wise choice of target ligand, and contributed to the authors success: the molecule offers several hydrogenbonding hooks, which helped to achieve high binding specificity in the protein, and the steroids rigid scaffold helped to maximize entropic contributions to binding. The authors were also able to gather information on what the binding site should look like from existing structures. Future work will, no doubt, implement ways to deal with flexible ligands and to design minimalistic binding sites in the absence of structural information. More power f ul computational methods would enable affinity maturation insilico rather than invitro, and would overcome the need for an existing scaffold as a starting point, by designing novel proteins around each target ligand15. Finally, molecular recognition could be coupled to functional activity, for example by using small-molecule binding to modulate protein activity. Giovanna Ghirlanda is in the Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA. e-mail: gghirlanda@asu.edu
1. Tinberg, C. E. et al. Nature http://dx.doi. org/10.1038/nature12443 (2013). 2. Gebauer, M. & Skerra, A. Meth. Enzymol. 503, 157188 (2012). 3. Gilbreth, R. N. & Koide, S. Curr. Opin. Struct. Biol. 22, 413420 (2012). 4. Jiang, L. et al. Science 319, 13871391 (2008). 5. Siegel, J. B. et al. Science 329, 309313 (2010). 6. Rthlisberger, D. et al. Nature 453, 190195 (2008). 7. Privett, H. K. et al. Proc. Natl Acad. Sci. USA 109, 37903795 (2012). 8. Korendovych, I. V. et al. Org. Lett. 12, 51425145 (2010). 9. Schreirer, B., Stumpp, C., Wiesner, S. & Hcker, B. Proc. Natl Acad. Sci. USA 106, 1849118496 (2009). 10. Metz, S. et al. Proc. Natl Acad. Sci. USA 108, 81948199 (2011). 11. Jeffrey, P. D. et al. Proc. Natl Acad. Sci. USA 90, 1031010314 (1993). 12. Korndrfer, I. P., Schlehuber, S. & Skerra, A. J. Mol. Biol. 330, 385396 (2003). 13. Fowler, D. M. et al. Nature Meth. 7, 741746 (2010). 14. Koide, S. & Sidhu, S. S. ACS Chem. Biol. 4, 325334 (2009). 15. Koga, N. et al. Nature 491, 222227 (2012).

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