N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

Abstract

Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.

Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers

“…Two genetically encoded fluorescent ligands, namely, AgTx2-GFP and ChTx-GFP, were used to bind assays as fluorescent probes. As shown by us earlier [14,27], chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP), namely, eGFP or TagRFP, fused with potassium channel toxins from scorpion venom (Tx) largely retain a high affinity of the natural toxins for the target Kv1 channels. These FP-Tx ligands have distinct advantages over fluorescent peptide blockers, which are obtained by the chemical conjugation of a peptide toxin with a fluorophore.…”

“…FP-Tx proteins are expressed in E. coli and purified from bacterial biomass with a high yield of about 100 mg per 1 L of bacterial culture; their purification procedure is rather simple and does not require any renaturation step. Introduced by means of bioengineering techniques of a fluorescent tag in a predetermined position (either N-or C-terminal) of the peptide toxin results in a more definite assessment of the binding activity of a fluorescent peptide [14].…”

“…Peptide toxins AgTx2 and ChTx, both tagged N-terminally with eGFP were used as fluorescent probes in the binding studies. Design and production of AgTx2-GFP, in which AgTx2 and eGFP moieties were separated by a 45-aa linker L1, was described earlier [14]. To obtain the analogous hybrid protein ChTx-GFP, a gene encoding AgTx2 in the previously obtained plasmid pET23d-GFP-L1-AgTx2 [14] was replaced by the ChTx coding sequence from pET23d-MalE-L1-ChTx, using KpnI/HindIII sites.…”

“…Design and production of AgTx2-GFP, in which AgTx2 and eGFP moieties were separated by a 45-aa linker L1, was described earlier [14]. To obtain the analogous hybrid protein ChTx-GFP, a gene encoding AgTx2 in the previously obtained plasmid pET23d-GFP-L1-AgTx2 [14] was replaced by the ChTx coding sequence from pET23d-MalE-L1-ChTx, using KpnI/HindIII sites. Correct cloning of the recombinant gene in the resulting plasmid pET23d-GFP-L1-ChTx was confirmed by sequencing both strands (Evrogen, Russia).…”

“…Hybrid protein ChTx-GFP was expressed and purified as described earlier [14]. Briefly, E. coli Rosetta-gami (DE3) pLysS cells were transformed with a plasmid pET23-GFP-L1-ChTx and cultivated at 37 • C in Terrific Broth (TB) supplied with 100 mg/L ampicillin, 15 mg/L kanamycin, 12.5 mg/L tetracycline and 34 mg/L chloramphenicol.…”