Split-protein systems: beyond binary protein-protein interactions

Abstract

It has been estimated that 650,000 protein-protein interactions exist in the human interactome (Stumpf et al., 2008), a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, Escherichia coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches.

Figure 1

Illustration of conditional split-protein reassembly. A generic split-protein system is shown where a functional protein is dissected into two inactive fragments, purple and yellow. The attachment of two interacting proteins or protein domains brings the inactive fragments into close proximity and overcomes the entropic cost of fragmentation. This leads to the reassembly or complementation of the fragments thus providing a direct readout for the partnership between the interacting domains. Crystal structures of representative proteins which have been shown to be amenable to interaction dependent reassembly, where the N-terminal and C-terminal fragments are shown as purple and yellow respectively.

Figure 2

Detection of macromolecules using split-protein reassembly via ternary complexation. a) Schematics of split-protein translation in cell-free translation systems such as rabbit reticulocyte lysate or wheat germ extract, where interacting domains of interest can be rapidly produced and interrogated. b) Detection of unmodified native proteins via ternary complexation using receptor or single-chain antibody fragments attached to split-luciferase. c) DNA and DNA modifications, such as methylation, can be detected using appropriate fusions of split luciferase and user defined DNA binding domains. The genome can be scanned at all CpG methylated sites for specific types of modifications resulting from DNA damage. d) A quaternary strategy for detection of RNA utilizing RNA detection domains such as argonaute and pumilio domains. The pentameric assembly allows targeting any ssRNA or ssDNA utilizing a zinc finger binding hairpin guide sequence. e) Co-factors such as iron-sulfur clusters detected via split-venus and apoglutaredoxin fusion pairs that dimerize upon binding 2Fe2S. e) Monitoring the dynamics of poly-ADP Ribose (PAR) levels subsequent to DNA damage through attached PAR targeted APLF domains.

Figure 3

New strategies for split-protein systems. a) Selective activation of caspases incorporating TEV cleavage sites using a small molecule rapamycin triggered reassembly of split-TEV protease. b) Screening kinase inhibitors utilizing a three-hybrid strategy using a new peptide-small molecule chemical inducer of dimerization wherein a potential kinase inhibitor can be identified by split-luciferase disassembly. c) An autoinhibited coiled-coil split-luciferase turn-on protease sensor wherein the autoinhibition is relieved in presence of any protease of interest leading to split-luciferase reassembly. d) An autoinhibited strategy for detection of small molecules using supramolecular building blocks.