Structural and functional analysis of Hydra Actinoporin-Like Toxin 1 (HALT-1)

Abstract

Actinoporins are a family of α-pore-forming toxins (α-PFTs) that have been identified in sea anemones. Recently, a freshwater Hydra Actinoporin-Like Toxin (HALT) gene family was found in Hydra magnipapillata. Unlike sea anemone actinoporins that use sphingomyelin as their main recognition target, the HALTs proteins may recognise alternative lipid molecules as their target. To unveil the structural insights into lipid preference of HALTs protein as compared to sea anemone actinoporins, we have determined the first crystal structure of actinoporin-like toxin, HALT-1 at 1.43 Å resolution with an acetylated lysine residue K76. Despite the overall structure of HALT-1 sharing a high structural similarity to sea anemone actinoporins, the atomic resolution structure revealed several unique structural features of HALT-1 that may influence the lipid preference and oligomerisation interface. The HALT-1 contains a RAG motif in place of the highly conserved RGD motif found in sea anemone actinoporins. The RAG motif contributed to a sharper β9-β10 turn, which may sway its oligomerisation interface in comparison to sea anemone actinoporins. In the lipid-binding region, the HALT-1 contains a shorter α2 helix and a longer α2-β9 loop due to deletion and subsequently an insertion of five amino acid residues in comparison to the sea anemone actinoporins. Structure comparison and molecular docking analysis further revealed that the HALT-1 lipid-binding site may favour sphingolipids with sulfate or phosphate head group more than the sphingomyelin. The structure of HALT-1 reported here provides a new insight for a better understanding of the evolution and lipid recognition mechanism of actinoporin.

Pore-forming moss protein bryoporin is structurally and mechanistically related to actinoporins from evolutionarily distant cnidarians

“…1A and C). Interestingly, while similar loops are not present in any known actinoporin structure, a loop of the same length at the equivalent position is present in Hydra actinoporin-like toxin 1 (HALT-1, PDB: 7EKZ) (54), with which bryoporin shares 25 % amino acid identity and additional 39 % J o u r n a l P r e -p r o o f similarity, with the RMSD of 1.53 Å. Unlike in HALT-1, where 2-helix is shorter for four amino acids, in bryoporin this helix is of the same length as in the sea anemone actinoporins (Fig.…”

Enhanced production of recombinant HALT-1 pore-forming toxin using two-step chromatographic procedure

“…Protein impurities with the size of 60-75 kDa were detected in the eluted fractions (Fig. 6A, lanes [5][6][7][8][9][10][11][12][13][14][15][16]. Eluted fractions of rHALT-1 were pooled, and buffer-exchanged in phosphate binding buffer and added to the column containing SP sepharose resins.…”

Enhanced production of recombinant HALT-1 pore-forming toxin using two-step chromatographic procedure

“…HALTs are members of the actinoporin family as they share approximately 30% amino acid sequence similarity with sea anemone actinoporins [7 , 8] . The structure of HALT-1 also highly resembles the actinoporins [9] , it has an amphipathic N -terminal α-helix, a cluster of aromatic amino acids in the central domain and a less conserved C -terminal domain [7 , 9] . HALT-1 is the most well-studied among all HALTs, and thus far it is known to be released by offensive nematocysts and used in the prey capture [6] .…”

“…Residual contaminating proteins from E. coli was considered negligible in the subsequent assays since their amounts present in the assays would be at least 1000-fold less than that of rHALT-1. It was not until recently that rHALT-1 was subjected to two rounds of purification, immobilized metal affinity chromatography (IMAC) coupled with size-exclusion chromatography (SEC) [9] . The choice of the column selected for size-exclusion chromatography was HiLoad Superdex 75 PG 16/600 (GE Healthcare, Chicago, USA) which has a bed height of 60 cm.…”