Programmable polyproteams built using twin peptide superglues
Edited by Samuel H. Gellman, University of Wisconsin–Madison, Madison, WI, and approved December 24, 2015 (received for review September 28, 2015)
Significance
Many biological events depend on proteins working together as a team. Here we establish how to program team formation, covalently linking protein modules step by step. We split a domain from Streptococcus pneumoniae to form a peptide and protein pair, SnoopTag and SnoopCatcher, which form an isopeptide bond when mixed together. SnoopTag/SnoopCatcher reacted with each other but not with an alternative peptide/protein pair, SpyTag/SpyCatcher. We formed polyprotein chains by alternating SpyTag reaction with SnoopTag reaction. Cellular signaling often relies on integrated activation of different receptors, so we built polyprotein teams to stimulate Death Receptor and Growth Factor receptors, finding an optimal combination for cell-death induction in cancer cells. Programmable “polyproteams” provide a simple route to investigate or harness biological teamwork.
Abstract
Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Here we achieved sequence-programmed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. SpyTag peptide is engineered to spontaneously form an isopeptide bond with SpyCatcher protein. By engineering the adhesin RrgA from Streptococcus pneumoniae, we developed the peptide SnoopTag, which formed a spontaneous isopeptide bond to its protein partner SnoopCatcher with >99% yield and no cross-reaction to SpyTag/SpyCatcher. Solid-phase attachment followed by sequential SpyTag or SnoopTag reaction between building-blocks enabled iterative extension. Linear, branched, and combinatorial polyproteins were synthesized, identifying optimal combinations of ligands against death receptors and growth factor receptors for cancer cell death signal activation. This simple and modular route to programmable “polyproteams” should enable exploration of a new area of biological space.
Data Availability
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. KU296973, KU296974, KU296975, KU296976, KU356870, KU361182, KU361183, KU500643, KU500644, KU500645, and KU500646).
Acknowledgments
We thank Chris Schoene (University of Oxford) for biotin-SpyCatcher, University of Oxford Department of Biochemistry Biophysical Facility for assistance, and Ario de Marco (University of Nova Gorica) for Erv1p and DsbC plasmids. Funding was provided by the Medical Research Council (G.V. and J.Y.), Merton College Oxford (G.V.), Sony (T.N.), the Ecole Normale Supérieure de Lyon (R.V.G.), the Royal Society (C.V.R.), and European Research Council Grant ERC-2013-CoG 615945-PeptidePadlock (to M.D.B. and M.H.).
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Cover image: Pictured is an aerial view of Chetro Ketl, one of the largest great houses at Chaco Canyon, New Mexico. Christopher H. Guiterman et al. found that nearly all of the timbers used to construct the Chaco great houses prior to 1020 CE came from the Zuni Mountains 85 kilometers south of Chaco Canyon, whereas most of the timbers after 1060 CE came from the Chuska Mountains, 75 kilometers to the west. The shift in timber source coincided with substantial great house construction during the florescence of the Chacoan system. See the article by Guiterman et al. on pages 1186–1190. Image courtesy of Adriel Heisey (photographer).
Classifications
Data Availability
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. KU296973, KU296974, KU296975, KU296976, KU356870, KU361182, KU361183, KU500643, KU500644, KU500645, and KU500646).
Submission history
Published online: January 19, 2016
Published in issue: February 2, 2016
Keywords
Acknowledgments
We thank Chris Schoene (University of Oxford) for biotin-SpyCatcher, University of Oxford Department of Biochemistry Biophysical Facility for assistance, and Ario de Marco (University of Nova Gorica) for Erv1p and DsbC plasmids. Funding was provided by the Medical Research Council (G.V. and J.Y.), Merton College Oxford (G.V.), Sony (T.N.), the Ecole Normale Supérieure de Lyon (R.V.G.), the Royal Society (C.V.R.), and European Research Council Grant ERC-2013-CoG 615945-PeptidePadlock (to M.D.B. and M.H.).
Notes
This article is a PNAS Direct Submission.
Authors
Competing Interests
Conflict of interest statement: M.H. is an inventor on patent EP2534484, which applies to spontaneous isopeptide bond formation to a peptide tag, and United Kingdom Patent Application No. 1509782.7, which applies to iterative synthesis through isopeptide bonds.
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