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The Kunitz-Type Protein ShPI-1 Inhibits Serine Proteases and Voltage-Gated Potassium Channels.
García-Fernández R
,
Peigneur S
,
Pons T
,
Alvarez C
,
González L
,
Chávez MA
,
Tytgat J
.
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The bovine pancreatic trypsin inhibitor (BPTI)-Kunitz-type protein ShPI-1 (UniProt: P31713) is the major protease inhibitor from the sea anemone Stichodactyla helianthus. This molecule is used in biotechnology and has biomedical potential related to its anti-parasitic effect. A pseudo wild-type variant, rShPI-1A, with additional residues at the N- and C-terminal, has a similar three-dimensional structure and comparable trypsin inhibition strength. Further insights into the structure-function relationship of rShPI-1A are required in order to obtain a better understanding of the mechanism of action of this sea anemone peptide. Using enzyme kinetics, we now investigated its activity against other serine proteases. Considering previous reports of bifunctional Kunitz-type proteins from anemones, we also studied the effect of rShPI-1A on voltage-gated potassium (Kv) channels. rShPI-1A binds Kv1.1, Kv1.2, and Kv1.6 channels with IC50 values in the nM range. Hence, ShPI-1 is the first member of the sea anemone type 2 potassium channel toxins family with tight-binding potency against several proteases and different Kv1 channels. In depth sequence analysis and structural comparison of ShPI-1 with similar protease inhibitors and Kv channel toxins showed apparent non-sequence conservation for known key residues. However, we detected two subtle patterns of coordinated amino acid substitutions flanking the conserved cysteine residues at the N- and C-terminal ends.
Figure 1. Protease inhibition curves using rShPI-1A. Fixed concentrations of each enzyme were mixed with increasing concentrations of rShPI-1A (displayed in nM). Each enzymatic activity was determined after adding the corresponding substrate (see Materials and Methods for experimental details). The fractional enzymatic activities (vi/v0) were calculated after measuring the initial reaction rates with (vi) and without inhibitor (v0). Each connecting line represents the best fits to the quadratic Morrison equation for tight binding inhibitors described in [21]. Apparent Ki values (Kiapp) were calculated by adjusting the experimental points to that equation and are shown here as the mean ± SE (n = 3).
Figure 2. Activity of rShPI-1A on ion channels expressed in X. laevis oocytes. Traces are representative of at least three independent experiments (n ≥ 3). The dotted line indicates the zero current level. The asterisk (*) distinguishes the steady-state current after application of 50 nM peptide.
Figure 3. Characterization of rShPI-1A activity on channel gating. Left panel: current-voltage relationship. Closed symbols are the control condition; open symbols are after application of 50 nM peptide. Right panel: concentration-response curve on Kv1.1 and Kv1.2 channels obtained by plotting the percentage of blocked current as a function of increasing toxin concentrations. All data represent at least three independent experiments (n ≥ 3) and are presented as the mean ± standard error.
Figure 4. Sequence alignment of Kv-channel blocking toxins. Residues reported as crucial in the activity toward Kv channels are underlined. Conserved Cys residues are in bold and (*) represent conserved residues. Positions buried to solvent or partially exposed are highlighted in gray. Residues with a negative charge are highlighted in red; positive charge (blue); polar uncharged (magenta); and hydrophobic (green). The numbering system in the corresponding UniProt entry (protein name and UniProt accession code) is used here. Continuous line boxes enclose the sequence regions around CysI and CysV-CysVI, which show an amino acid substitution pattern in most of these proteins. A discontinuous line box encloses key residues around CysII. Additional data about toxins included in this alignment are provided in Table 3 and Table 4. A list of the ShPI-1 interatomic contacts and the accessible surface area (ASA) relative to the residue in a vacuum is provided in the Supplementary File 1.
Figure 5. Structure-based sequence alignment of Kv-channel blocking toxins. Uppercase letters represent the equivalent position in the 3D superimposition. Continuous line boxes enclose those regions that superimpose well among toxins sharing the BPTI-Kunitz fold and those from ShK-type and scorpion-type families, also active against Kv channels. Information about their respective secondary structure elements, helix (h) and β-sheet (e), is shown over each sequence, based on ShPI-1 as representative of all BPTI-Kunitz-type proteins and the toxins ShK, BgK and ChTx. Information about toxin family, including the Pfam database (PF) code, is shown at the right. Conserved Cys residues are in bold, and positions buried to solvent or partially exposed are highlighted in gray. Residues with a negative charge are highlighted in red; positive charge (blue); polar uncharged (magenta); and hydrophobic (green).
Figure 6. Surface electrostatic potential representation of toxins. The surface is colored according to the electrostatic potential: negative regions (in red), positive regions (in blue) and neutral regions (in gray). The orientation of the surface electrostatic potentials is the same as that in the ribbon representation at the bottom right. The list of PDB codes for toxins is as follows: ShPI-1 (3M7Q); LmKTT-1a (2M01); HWTX-XI (2JOT); Conk-S1 (1Y62); α-DTX (1DTX); DTX-K (1DTK); DTX-I (1DEM); BgK (1BGK); ShK (4LFS); and ChTX (1BAH). We also provided a color intensity scale to better represent the electrostatic potential. BPTI-Kunitz-type toxins are displayed in the first two lines. These figures were prepared with the PyMOL Molecular Graphics System, Schrödinger, LLC.
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