Genomic and structural characterization of Kunitz-type peptide LmKTT-1a highlights diversity and evolution of scorpion potassium channel toxins

Abstract

Background: Recently, a new subfamily of long-chain toxins with a Kunitz-type fold was found in scorpion venom glands. Functionally, these toxins inhibit protease activity and block potassium channels. However, the genomic organization and three-dimensional (3-D) structure of this kind of scorpion toxin has not been reported.

Principal findings: Here, we characterized the genomic organization and 3-D nuclear magnetic resonance structure of the scorpion Kunitz-type toxin, LmKTT-1a, which has a unique cysteine pattern. The LmKTT-1a gene contained three exons, which were interrupted by two introns located in the mature peptide region. Despite little similarity to other Kunitz-type toxins and a unique pattern of disulfide bridges, LmKTT-1a possessed a conserved Kunitz-type structural fold with one α-helix and two β-sheets. Comparison of the genomic organization, 3-D structure, and functional data of known toxins from the α-KTx, β-KTx, γ-KTx, and κ-KTx subfamily suggested that scorpion Kunitz-type potassium channel toxins might have evolved from a new ancestor that is completely different from the common ancestor of scorpion toxins with a CSα/β fold. Thus, these analyses provide evidence of a new scorpion potassium channel toxin subfamily, which we have named δ-KTx.

Conclusions/significance: Our results highlight the genomic, structural, and evolutionary diversity of scorpion potassium channel toxins. These findings may accelerate the design and development of diagnostic and therapeutic peptide agents for human potassium channelopathies.

Figure 1. Amino acid sequence alignment of LmKTT-1a with Kunitz-type toxins from other venomous animals.

Representative Kunitz-type toxins are LmKTT-1a from scorpion, Conkunitzin-S1 (PDB Code: 1Y62) from conus, APEKTx1 (PDB Code: 1WQK) from sea anemone, α-DTX (PDB Code: 1DTX) from snake, and HWTXI-XI (PDB Code: 2JOT) from spider. The known disulfide bridges are labeled in black lines. The red dotted line suggests a possible new disulfide bridge.

Figure 2. Genomic organization of scorpion Kunitz-type toxin, LmKTT-1a.

(A) The LmKTT-1a gene. The signal peptide sequence predicted from the nucleotide sequence is underlined. The putative polyadenylation signal (AATAAA) is underlined twice. (B) The gene structure of LmKTT-1a. The signal peptide (SP), mature peptide (MP), 5′-UTR, and 3′-UTR non-coding regions are shown. Introns are designated by triangles.

Figure 3. Inhibition of Kv1 potassium channel activity by LmKTT-1a.

(A) Current traces of the Kv1.1 channel in the absence (control) or presence of 1 µM LmKTT-1a. (B) Current traces of the Kv1.2 channel in the absence (control) or presence of 1 µM LmKTT-1a. (C) Current traces of the Kv1.3 channel in the absence (control) or presence of 1 µM LmKTT-1a. (D) Concentration-dependent inhibition of Kv1.3 channel currents by LmKTT-1a. Data represent the mean ± S.D. of at least three experiments.

Figure 4. NMR solution structure of LmKTT-1a.

A) Superposition of the 20 structures with lowest total energy. (B) Ribbon presentation of the backbone of LmKTT-1a from scorpion. (C) Ribbon presentation of the backbone of ConK-S1 from snail. (D) Ribbon presentation of the backbone of α-dendrotoxin from snake. (E) Ribbon presentation of the backbone of HWTX-XI from spider. (F) Ribbon presentation of the backbone of APEKTX1 from sea anemone.

Figure 5. Functional evaluation of the unique disulfide bridge, Cys51–Cys59, in LmKTT-1a. (

A) A mutant LmKTT-1a-C51A/C59A lacking the unique disulfide bridge Cys51–Cys59 was designed from LmKTT-1a. Ki values for trypsin are labeled in bold font. (B) Current traces in the absence (control) or presence of 1 µM LmKTT-1a-C51A/C59A and LmKTT-1a. (C) Structural stability of the LmKTT-1a mutant, LmKTT-1a-C51A/C59A.

Figure 6. Diverse structural fold of scorpion potassium channel toxins.

(A) Ribbon presentation of the backbone of KTX from the α-KTX subfamily, which has a CSα/β fold. (B) Ribbon presentation of the backbone of OmTx1 from the κ-KTX subfamily, which has a CSα/α fold. (C) Ribbon presentation of the backbone of LmKTT-1a from a new subfamily with a Kunitz-type fold.

Figure 7. Comparison of the gene structures of representative scorpion potassium channel toxins.

The gene structures of scorpion potassium channel toxins BmKTX from the α-KTX subfamily, which has a CSα/β fold, TtrKIK and BmTXKβ2 from the β-KTX subfamily, which has a CSα/β fold, BmKK7 from the γ-KTX subfamily, which has a CSα/β fold, HeTx203 from the κ-KTX subfamily, which has a CSα/α fold, and LmKTT-1a and BmKTT-2 from δ-KTX, the new subfamily of Kunitz-type fold toxins. The signal peptide (SP), propeptide (PP), mature peptide (MP), 5′-UTR, and 3′-UTR non-coding regions are shown. Introns are designated by triangles.

Figure 8. Molecular diversity and classification of scorpion potassium channel toxins.

Representative potassium channel toxins from the α-KTxs, β-KTxs, γ-KTxs, κ-KTxs, and δ-KTxs subfamily are listed. All members from the δ-KTxs subfamily with a Kunitz-type fold are shown.

Figure 9. Schematic diagram of the evolution of scorpion potassium channel toxins.

Three putative ancestors were recruited from scorpion proteins to generate diverse potassium channel toxins from five different subfamilies (α-KTxs, β-KTxs, γ-KTxs, κ-KTxs, and δ-KTxs) with three different structural folds (CSα/β, CSα/β, and Kunitz-type).