Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes

Abstract

Live cell imaging is a powerful method for studying protein dynamics at the cell surface, but conventional probes, such as antibodies and fluorescent ligands, are bulky, interfere with protein function 1,2 , or dissociate after internalization 3,4 . To overcome these limitations, we developed a method to covalently tag any cell surface protein with any chemical probe with remarkable specificity. Through rational design, we re-directed a microbial lipoic acid ligase (LplA) 5 to specifically ligate an alkyl azide to an engineered LplA acceptor peptide (LAP) tag. The alkyl azide is then selectively derivatized with a cyclooctyne 6 conjugated to any probe of interest. We demonstrate the utility of this method by first labeling LAP fusion proteins expressed on the surface of living mammalian cells with Cy3, Alexa Fluor 568, and biotin. Next, we combined LAP-tagging with our previously reported tagging method 7,8 to simultaneously monitor the dynamics of two receptors, co-expressed in the same cell, with different fluorophores. Using a wound-healing assay, we found that while the LDL receptor maintains a uniform distribution on the cell surface, the ephrin receptor EphA3 is polarized to the leading edge. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.Fluorescent labeling of cell surface proteins enables imaging of the trafficking and function of receptors, channels, and transporters. Many protein labeling methods have been developed in recent years 9 , but none currently allows the covalent attachment of small fluorophores of any structure onto cell surface proteins modified only by a small peptide tag, with short labeling times and with extremely high specificity over a wide range of expression levels and labeling conditions. To address this shortcoming, we developed a new protein labeling method based on the E. coli enzyme lipoic acid ligase (LplA) 5 . In E. coli, LplA catalyzes the ATP-dependent covalent ligation of lipoic acid to one of three proteins involved in oxidative metabolism (E2p, E2o, and H-protein 5 ) (Fig. 1a, top). LplA naturally exhibits extremely high sequence specificity, but previous work showing that the enzyme accepts octanoic acid, 6-thio-octanoic acid, and selenolipoic acid in place of lipoic acid 5 suggest that the small-molecule binding site has considerable plasticity. To harness LplA for fluorescent labeling, we re-engineered the system in three stages. First, through synthesis and testing of ten different substrate analogs, we discovered an alkyl azide substrate that can be efficiently used by LplA in place of lipoic acid. Once ligated to the target protein, the azide functional group can be selectively derivatized with any fluorescent probe conjugated to a cyclooctyne reaction partner 6 (Figure 1a). Second, to create a minimally invasive tag to direct the ligation of the alkyl azide, we engineered, through iterative cycles of rational design, a 22-amino acid replacement...

Chemical tags: Applications in live cell fluorescence imaging

“…LplA is structurally related to biotin ligase and shows mechanistic parallels to BirA. Similar to BirA, the Ting lab optimized LplA for substrates carrying an azide functionality [52]. By mutational analysis, the residue W37 was identified to be the most important for expanding the substrate specificity.…”

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

“…LplA mutants with an N-terminal His 6 tag and E2p lipoyl domain were purified as previously reported (42). LplA was dialyzed against and stored in 20 mM Tris·HCl, pH 7.5, containing 10% (vol/vol) glycerol and 1 mM DTT, at -80°C.…”

“…Both H 2 O and acetonitrile solvents were supplemented with 0.1% formic acid. Peptide and resorufin-peptide adducts were quantified by integrating their UV absorption peaks at 210 nm, with correction from resorufin absorption using a previously reported method (42). LAP and LAP-F peptides were synthesized and purified to >95% purity by GenScript.…”

Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

“…), [14][15][16] or are recognized by enzymes that can transfer a separate substrate moiety, which is fluorescent or can be orthogonally modified (biotin ligase, sortase, lipoic acid ligase, formylglycine-generating enzyme) have emerged as powerful strategies. [17][18][19][20] A complement to the aforementioned approaches is the application of fluorescent α-amino acid analogues, which have the advantage of being relatively non-perturbing replacements for the native encoded residues, thereby maintaining the overall native structure of a target peptide or protein. In addition, the modularity of α-amino acid building blocks makes them versatile components in the protein assembly toolbox and thus, once syntheses and methods for peptide and protein incorporation are developed new applications can be readily adopted.…”