Palladium-Responsive Peptides

An extended version of the following text has appeared in the Nature Portfolio Behind the Paper series: Controlling Oncogenic KRAS Signaling Pathways with a Palladium-Responsive Peptide.

The original article, 1. S. Learte-Aymamí, P. Martin-Malpartida, L. Roldán-Martín, G. Sciortino, J. R. Couceiro, J.-D. Maréchal, M. J. Macias, J. L. Mascareñas, M. E. Vázquez, Controlling oncogenic KRAS signaling pathways with a Palladium-responsive peptide.
Communications Chemistry. 5, 1–9 (2022), can be found here.
In the spring of 1948, while playing with a strip of paper in which he had drawn a polypeptide chain, Linus Pauling built the first model of an α-helix. This initial insight on the three-dimensional arrangement of proteins led him, together with Robert B. Corey and Herman R. Branson, to a foundational publication in structural biology, some say as important as that by Watson and Crick describing the DNA duplex, in which they predicted the atomic structure of the α-helix 1. Over the years, researchers have realized that α-helices are not only one of the fundamental protein building blocks but also frequent mediators in protein-protein and protein-DNA interactions that regulate key signaling pathways. It has been estimated that roughly 60% of all protein complexes feature α-helical interfaces 2. α-helices are, thus, a key ingredient of the language that proteins use to communicate, and so they have become a hot target for the development of new protein-protein interaction inhibitors. But developing such α-helical inhibitors is not straightforward because α-helices are delicate structures and short peptide sequences do not usually maintain their helical structure when extracted from their parent protein. This can be understood going back to Pauling’s folded paper model, which you would need to hold together with your hands or a piece of tape, otherwise, the helix unrolls and the peptide is straightened out. Much like it, at the molecular level, α-helices are, at best, marginally stable structures because the energy required to fold them into the correct helical arrangement, what is known as the entropic cost, is hardly compensated by the little energy gained upon folding, the enthalpic term. Unfolded helices are pretty much useless: they do not present their functional groups in the right place to create a binding site, and the energy required to fold them, taxes the formation of their complexes so much that the binding affinity would be too low.
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One of the most successful strategies to develop α-helical inhibitors has been the preorganization of the α-helical conformation to reduce the entropic term. This can be done by introducing crosslinks between side chains in the same face of the α-helix, which are typically known as (i,i+4) and (i,i+7) positions according to the number of amino acid residues between the crosslink 3. Examples of this strategy include lactam bridges between the side chains of Lys and Asp residues 4, salt bridges between charged Glu and Lys residues 5, and metal ion coordination with appropriately installed His residues in adjacent helical turns 6. Currently, most crosslinked peptidomimetics rely on hydrocarbon linkers formed by ruthenium-catalyzed ring-closing metathesis (RCM) reactions between unnatural alkenyl amino acids 7. In contrast to these constitutively active inhibitors, locked into stable α-helical conformations by permanent clamps, stimuli-responsive peptides that change their affinity in response to an external input could offer new opportunities in basic research, as well as for spatiotemporal controlled therapeutic applications. Thus, going beyond the classic metal-stabilized α-helices, and considering our previous studies in the field of DNA binding 8, we hypothesized that metal chelation could be applied for the development of switchable α-helical peptides that modify their folding and binding affinity in response to an external stimulus, namely, the presence of a Pd(II).

Toroidal proteins for nanotechnological applications

Proteins are outstanding platforms for the construction of functional nanostructures with applications in biomedicine, biotechnology or nanomaterials 1. Toroidal proteins, in particular, have found widespread applications for the bottom-up fabrication of nanotubes 2, and for the templated synthesis and three-dimensional organization of metal nanoparticles 3. Nevertheless, the reported examples rely on the engineering and self-assembly of large proteins (e.g., SP1, 148.8 KDa; TRAP, 92 KDa), which are only accessible through recombinant protein expression systems, and offer little synthetic flexibility for the introduction of extrinsic functionalities.

We envisioned that
we could replicate the functional and architectural complexity of large ring proteins using synthetically accessible peptides that self-assemble into toroidal oligomers. With this aim in mind, we screened the pdb database and selected for our study the viral protein gp23.1, which is a small three-helix bundle composed by 50 residues 4. Importantly, gp23.1 forms a hexamer in solution, as revealed by light scattering measurements, and its structure has been solved by X-ray crystallography. Noteworthy, gp23.1 exhibits a toroidal structure that is about 60 Å in diameter, with an 20 Å-wide central channel delimited by the six α1 helices (Fig. 1), whereas α2 and α3 helices define the outside surface of the hexamer.
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The small size of the gp23.1 monomer puts it within the reach of standard solid-phase peptide synthesis methods, which allow the straightforward preparation of new sequences, including those with artificial functionalities, as well as the implementation of powerful postsynthetic modifications . This sinthetic availability, combined with the predictable oligomerization of these chemically synthesized monomers, makes of gp23.1 a potentially valuable platform for bottom-up nanotechnological applications.

As a proof-of-concept application of inner surface engineering of gp23.1, we introduced the D12C mutation in the α1 helix. Self-assembly of this mutant would place six thiol groups in the interior of the hexamer central pore, thus generating an epitope for the templated growth of gold nanoclusters (AuNCs) of uniform size, fitting the interior cavity of the gp23.1 ring.3b, Also, to avoid potential oxidation problems during the acidic deprotection/cleavage step and peptide handling, our initial target sequence replaced the Met42 residue by the isosteric norleucine.
  1. Pauling, L., Corey, R.B., and Branson, H.R. (1951). The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. Proc. Natl. Acad. Sci. USA 37, 205–211.
  2. Jochim, A.L., and Arora, P.S. (2010). Systematic analysis of helical protein interfaces reveals targets for synthetic inhibitors. ACS Chem. Biol. 5, 919–923.
  3. A) de Araujo, A.D., Hoang, H.N., Kok, W.M., Diness, F., Gupta, P., Hill, T.A., Driver, R.W., Price, D.A., Liras, S., and Fairlie, D.P. (2014). Comparative α-helicity of cyclic pentapeptides in water. Angew. Chem. Int. Ed. 53, 6965–6969; b) Lau, Y.H., de Andrade, P., Wu, Y., and Spring, D.R. (2015). Peptide stapling techniques based on different macrocyclisation chemistries. Chem. Soc. Rev. 44, 91–102.
  4. a) Hoang, H.N., Wu, C., Hill, T.A., Dantas de Araujo, A., Bernhardt, P.V., Liu, L., and Fairlie, D.P. (2019). A Novel Long-Range n to π* Interaction Secures the Smallest known α-Helix in Water. Angew. Chem. Int. Ed. 58, 18873–18877; b) Shepherd, N.E., Hoang, H.N., Abbenante, G., and Fairlie, D.P. (2005). Single turn peptide α helices with exceptional stability in water. J. Am. Chem. Soc. 127, 2974–2983.
  5. Marqusee, S., and Baldwin, R.L. (1987). Helix stabilization by Glu-...Lys+ salt bridges in short peptides of de novo design. Proc. Natl. Acad. Sci. USA 84, 8898–8902.
  6. a) Ghadiri, M.R., and Choi, C. (1990). Secondary structure nucleation in peptides. Transition metal ion stabilized α-helices. J. Am. Chem. Soc. 112, 1630–1632; b) Ghadiri, M.R., and Fernholz, A.K. (1990). Peptide architecture. Design of stable α-helical metallopeptides via a novel exchange-inert ruthenium(III) complex. J. Am. Chem. Soc. 112, 9633–9635; c) Kelso, M.J., Hoang, H.N., Appleton, T.G., and Fairlie, D.P. (2000). The First Solution Structure of a Single α-Helical Turn. A Pentapeptide α-Helix Stabilized by a Metal Clip. J. Am. Chem. Soc. 122, 10488–10489; d) Ma, M.T., Hoang, H.N., Scully, C.C.G., Appleton, T.G., and Fairlie, D.P. (2009). Metal clips that induce unstructured pentapeptides to be α-helical in water. J. Am. Chem. Soc. 131, 4505–4512.
  7. Cromm, P.M., Spiegel, J., and Grossmann, T.N. (2015). Hydrocarbon stapled peptides as modulators of biological function. ACS Chem. Biol. 10, 1362–1375.
  8. a) Gómez-González, J., Pérez, Y., Sciortino, G., Roldan-Martín, L., Martínez-Costas, J., Maréchal, J.-D., Alfonso, I., Vázquez López, M., and Vázquez, M.E. (2021). Dynamic Stereoselection of Peptide Helicates and Their Selective Labeling of DNA Replication Foci in Cells. Angew. Chem. Int. Ed. 60, 8859–8866; b) Learte-Aymamí, S., Curado, N., Rodríguez, J., Vázquez, M.E., and Mascareñas, J.L. (2017). Metal-Dependent DNA Recognition and Cell Internalization of Designed, Basic Peptides. J. Am. Chem. Soc. 139, 16188–16193; c) Rodríguez, J., Mosquera, J., Vázquez, M.E., and Mascareñas, J.L. (2016a). Nickel-Promoted Recognition of Long DNA Sites by Designed Peptide Derivatives. Chem. Eur. J. 22, 13474–13477; d) Rodríguez, J., Mosquera, J., García-Fandiño, R., Vázquez, M.E., and Mascareñas, J.L. (2016b). A designed DNA binding motif that recognizes extended sites and spans two adjacent major grooves. Chem. Sci. 7, 3298–3303.

  9. a) G. C. Pugh, J. R. Burns and S. Howorka, Nat. Rev. Chem., 2018, 2, 113; b) D. J. Glover and D. S. Clark, ACS Cent Sci, 2016, 2, 438; c) F. Lapenta, J. Aupič, Ž. Strmšek and R. Jerala, Chem. Soc. Rev., 2018, 47, 3530; d) R. V. Ulijn and R. Jerala, Chem. Soc. Rev., 2018, 47, 3391; e) D. Sánchez-deAlcázar, S. H. Mejías, K. Erazo, B. Sot and A. L. Cortajarena, J. Struct. Biol., 2018, 201, 118.
  10. a) G. Rao, Y. Fu, N. Li, J. Yin, J. Zhang, M. Wang, Z. Hu and S. Cao, ACS Appl. Mater. Interfaces, 2018, 10, 25135; b) T. K. Nguyen, H. Negishi, S. Abe and T. Ueno, Chem. Sci., 2019, 10, 1046; c) F. F. Miranda, K. Iwasaki, S. Akashi, K. Sumitomo, M. Kobayashi, I. Yamashita, J. R. H. Tame and J. G. Heddle, Small, 2009, 5, 2077; d) T. Sendai, S. Biswas and T. Aida, J. Am. Chem. Soc., 2013, 135, 11509.
  11. a) O. K. Zahr and A. S. Blum, Nano Lett., 2012, 12, 629–633; b) A. Schreiber, M. C. Huber, H. Cölfen and S. M. Schiller, Nat. Commun., 2015, 6, 6705; c) A. D. Malay, J. G. Heddle, S. Tomita, K. Iwasaki, N. Miyazaki, K. Sumitomo, H. Yanagi, I. Yamashita and Y. Uraoka, Nano Lett., 2012, 12, 2056.
  12. D. Veesler, S. Blangy, J. Lichière, M. Ortiz-Lombardía, P. Tavares, V. Campanacci and C. Cambillau, Protein Sci., 2010, 19, 1812.