Nonproteinogenic Deep Mutational Scanning of Linear and Cyclic Peptides

Nonproteinogenic deep mutational scanning of linear and cyclic peptides Joseph M. Rogersa, Toby Passiouraa, and Hiroaki Sugaa,b,1 aDepartment of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan; and bCore Research for Evolutionary Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan Edited by David Baker, University of Washington, Seattle, WA, and approved September 18, 2018 (received for review June 10, 2018) High-resolution structure–activity analysis of polypeptides re- mutants that can be constructed (18). Moreover, it is possible to quires amino acid structures that are not present in the universal combine parallel peptide synthesis with measures of function genetic code. Examination of peptide and protein interactions (19). However, these approaches cannot construct peptide li- with this resolution has been limited by the need to individually braries with the sequence length and numbers that deep muta- synthesize and test peptides containing nonproteinogenic amino tional scanning can, which, at its core, uses high-fidelity nucleic acids. We describe a method to scan entire peptide sequences with acid-directed synthesis of polypeptides by the ribosome. multiple nonproteinogenic amino acids and, in parallel, determine Ribosomal synthesis (i.e., translation) can be manipulated to the thermodynamics of binding to a partner protein. By coupling include nonproteinogenic amino acids (20). In vitro genetic code genetic code reprogramming to deep mutational scanning, any reprogramming is particularly versatile, allowing for the in- number of amino acids can be exhaustively substituted into pep- corporation of amino acids with diverse chemical structures (21). tides, and single experiments can return all free energy changes of Flexizymes, flexible tRNA-acylation ribozymes, can load almost binding. We validate this approach by scanning two model any (ester-activated) amino acid onto any tRNA, and these protein-binding peptides with 21 diverse nonproteinogenic amino loaded tRNAs can be added to reconstituted in vitro translation – acids. Dense structure activity maps were produced at the resolu- systems to replace proteinogenic amino acids in the genetic code tion of single aliphatic atom insertions and deletions. This permits (Fig. 1A and SI Appendix, Fig. S1). These “reprogrammed” ge- rapid interrogation of interaction interfaces, as well as optimization netic codes allow for the one-pot synthesis of trillions of unique, of affinity, fine-tuning of physical properties, and systematic assess- nonproteinogenic amino acid-containing peptides derived from a ment of nonproteinogenic amino acids in binding and folding. pool of mRNA sequences. Members of these peptide libraries can be coupled to their encoding mRNA/cDNA, allowing for the BH3 domains | noncanonical amino acids | structure–activity relationships | isolation of functional, highly nonproteinogenic peptides; most macrocyclic peptides | intrinsically disordered proteins notably, de novo macrocyclic peptides (22) from the random nonstandard peptide integrated discovery (RaPID) system (23– — he chemical structure of a polypeptide determines its activity 26). Here, we use flexizyme-based genetic code reprogramming Tincluding any folding or binding. Specific changes to chem- to extend the reach of deep mutational scanning to examine any ical structure (mutants) can be analyzed in large numbers (1). number of nonproteinogenic amino acids in peptide binding Deep mutational scanning methods, in particular, allow for the and folding. analysis of many thousands of mutants (2): Mutant proteins or peptides are each coupled to their encoding DNA, libraries of pooled Significance mutants are sorted for activity, mutants are counted via deep sequencing, and each mutant is scored. The throughput of these experiments is sufficient for exhaustive saturation mutagenesis; that is, The 20 proteinogenic amino acids have physicochemical prop- testing all proteinogenic amino acids at all positions in a sequence erties that allow peptides and proteins to fold and bind. and returning all effects on folding (3–5), binding (2, 5–8), or function However, there are numerous unnatural, nonproteinogenic (9). Extensive structure–activity maps are produced, but these amino acids that may be equally good, or even better, at methods are currently limited to the chemistry accessible within the folding and binding. Exploration of these alternative peptide universal genetic code—the 20 proteinogenic amino acids. building blocks has been limited by slow, one-at-a-time syn- However, extending mutagenesis to include nonproteinoge- thesis and testing. We describe how, in a single experiment, nic amino acids offers many advantages. The larger range of multiple nonproteinogenic amino acids can be trialed at all chemical structures allows for a finer dissection, or optimization, positions in a peptide sequence, with thousands of modifica- of molecular interactions, down to single aliphatic carbon in- tions tested in parallel. This permits detailed analysis of how sertions and deletions or functional group substitutions (10, 11). chemical structure relates to function and allows for systematic Certain nonproteinogenic amino acids can also improve the comparisons of proteinogenic and nonproteinogenic chemistry. Such analysis can guide the improvement of drug-candidate otherwise poor in vivo stability of short peptides and/or reduce peptides, including the therapeutically promising class of cyclic the excessive polarity that prevents peptides crossing cell mem- peptides. branes (12, 13). Indeed, nonproteinogenic amino acids are abundant in peptide natural products (14) and peptides modified Author contributions: J.M.R. designed research; J.M.R. and T.P. performed research; J.M.R. for in vivo use (10, 11). Nonproteinogenic amino acid muta- and T.P. contributed new reagents/analytic tools; J.M.R. analyzed data; and J.M.R., T.P., CHEMISTRY genesis can also address longstanding questions about the fitness and H.S. wrote the paper. of the universal genetic code and its collection of amino acids The authors declare no conflict of interest. relative to plausible prebiotic alternatives (15), as well as guide This article is a PNAS Direct Submission. development of synthetic polymers with the ability to fold (i.e., This open access article is distributed under Creative Commons Attribution-NonCommercial- foldamers) (16, 17). NoDerivatives License 4.0 (CC BY-NC-ND). Exploration of nonproteinogenic amino acid mutants has been 1To whom correspondence should be addressed. Email: [email protected]. previously limited by the need to chemically synthesize and an- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. alyze peptides individually (10, 11). Recent advances in peptide 1073/pnas.1809901115/-/DCSupplemental. BIOPHYSICS AND synthesis have extended the numbers of nonproteinogenic Published online October 9, 2018. COMPUTATIONAL BIOLOGY www.pnas.org/cgi/doi/10.1073/pnas.1809901115 PNAS | October 23, 2018 | vol. 115 | no. 43 | 10959–10964 Downloaded by guest on September 24, 2021 M144A (henceforth referred to as the wild-type); this mutation A amino acid tRNA ribosome peptide-mRNA DNA peptide-cDNA barcode prevents oligomerization of PUMA peptides at high (micromo- lar) concentrations. Each peptide was covalently attached to its 1 CME H2N encoding mRNA via a puromycin linker, and the pool of pep- O tide–mRNA fusions was reverse transcribed into noncovalent A CME cDNA complexes using barcoded DNA primers (Fig. 1 ) (32). 2 N H O Barcoding, which encoded the reprogrammed genetic codes 3 DBE H2N themselves, permitted an outsized 41 amino acid alphabet. O 4 DBE The resulting diverse PUMA peptide library was incubated H2N O with immobilized MCL1 and washed, and the bound fraction was 5 DBE recovered (Fig. 1B). Populations before and after binding were H2N O enumerated by deep sequencing, and enrichment scores for B C E C OH OH OH OH OH binding ( ) were calculated for every mutant (2) (Fig. 1 ). To H N N H2N H2N H2N 2 H O O O O O validate this approach, previously reported KD/ΔΔG values (30) were compared with E scores, which revealed that E is a smooth te reS e s r psA n laV ueL ryT sy ylG ulG n grA orP s p alA l iH y sA hP h r e lI C E T G L T C M function of MCL1 binding affinity (Fig. 2 ). To validate that is a function of binding for nonproteinogenic mutants, we chemically synthesized additional PUMA mutants and measured output SI Appendix Sequence binding to MCL1 ( , Figs. S3 and S4 and Table S1). input For synthetic convenience, the PUMA peptides in this collection ΔΔ ei = Foutput,i / Finput,i = f(KD) E Ei = ei / ewild-type = f( G) were 27 amino acids in length, shorter than the 35-aa peptides used in previous studies (29, 30), and ΔΔG values were calcu- Fig. 1. Nonproteinogenic deep mutational scanning. (A) Essentially any lated relative to the binding of an equivalent 27-aa wild-type. nonproteinogenic amino acid [activated by cyanomethyl ester (CME) or The link between ΔΔG and E is maintained for this collection dinitrobenzyl ester (DBE)] can be loaded onto tRNA by flexizymes and de- and, importantly, the proteinogenic and nonproteinogenic mu- livered to the ribosome for use during in vitro translation. Translation of a tant data overlay (Fig. 2C). site-saturation mutagenesis mRNA library using genetic code reprogram- E scores alone are sufficient for analysis of deep mutational ming allows for a nonproteinogenic

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