Interaction of sea anemone Heteractis crispa Kunitz type polypeptides with pain vanilloid receptor TRPV1: In silico investigation

Abstract

Using methods of molecular biology we defined the structures of the 31 sea anemone Heteractis crispa genes encoding polypeptides which are structurally homologous to the Kunitz proteinase inhibitor family. Identified amino acid sequences have point residue substitutions, high degree of homology with sequences of known H. crispa Kunitz family members, and represent a combinatorial library of polypeptides. We generated their three-dimensional structures by homologous modeling methods. Analysis of their molecular electrostatic potential enabled us to divide given polypeptides into three clusters. One of them includes polypeptides APHC1, APHC2 and APHC3, which were earlier shown to possess a unique property of inhibiting of the pain vanilloid receptor TRPV1 in vitro and providing the analgesic effects in vivo in addition to their trypsin inhibitory activity. Molecular docking made possible establishing the spatial structure of the complexes, the nature of the polypeptides binding with TRPV1, as well as functionally important structural elements involved in the complex formation. Structural models have enabled us to propose a hypothesis contributing to understanding the APHC1-3 impact mechanism for the pain signals transduction by TRPV1: apparently, there is an increase of the receptor relaxation time resulted in binding of its two chains with the polypeptide molecule, which disrupt the functioning of the TRPV1 and leads to partial inhibition of signal transduction in electrophysiological experiments.

Kunitz-Type Peptide HCRG21 from the Sea Anemone Heteractis crispa Is a Full Antagonist of the TRPV1 Receptor

“…However, one unique peptide, HCRG21, has Thr14 at this position, similar to the representatives of so named “analgesic cluster” of HCGS peptide subfamily have. This cluster is composed of analgesic peptides APHC1–APHC3 which are also known as modulators of the TRPV1 receptor [28,30,31,32]. HCRG21 shares 82% to 95% of identity with APHC1–APHC3, as well as with Jn-IV, InhVJ, and some HCGS peptides deduced by the PCR-based cloning technique (HCGS1.10, HCGS1.34, HCGS1.36, HCGS1.26, and HCGS2.3) (Figure 1a) [11].…”

“…Previously, on the basis of experimental data [28] and computer modeling [32], it was suggested that the most functionally important residues are Glu6, Thr14, Val31, Glu38, Arg48, and Arg51 in APHC1. All these residues are conserved between APHC1 and HCRG21, except Val31, which is replaced by a Pro in HCRG21.…”

“…According to this model, a modulating effect of the peptide can be explained by its interaction with multiple (two or more) subunits of the regulatory receptor domain, which prevents its free transition from one state to another, and apparently extends the receptor relaxation time. Analysis of the architecture of the HCRG21 complex with the intracellular part of the receptor, derived from APHC1–TRPV1 complex [32] by homology docking and MD simulations have revealed that 26 HCRG21 residues, including Thr14, Tyr16 and Phe17, differing peptides of “analgesic cluster” (Figure 1b) from other H. crispa Kunitz-type peptides, are involved into hydrogen bonding and/or electrostatic interactions as well as π-interactions with all receptor subunits (in total 57 interactions, Table S2). …”

“…So, it remains unclear why the Thr residue at P1 position is of crucial importance for peptides of H. crispa “analgesic cluster” (Figure 1b). It should be also noted that peptides of this cluster unlike other members of HCGS and HCRG subfamilies have a very small net positive charge of the molecule (in range from 0 to +2) [32]. Moreover, they are characterized by a point distribution of the MEP [11,32].…”

“…It was also found that three H. crispa serine protease inhibitors, APHC1, APHC2, and APHC3, possess an analgesic activity [28,29,30,31] by exhibiting an inhibitory activity against the pain receptor TRPV1 [28,32]. They are the first sea anemone peptide characterized as TRPV1 modulators.…”